Des jeunes, acteurs de leur avenir

Lowest price furosemide

A still lowest price furosemide unanswered question is https://www.epide.fr/buy-furosemide-online-without-a-prescription/ what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower lowest price furosemide mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells.

Conversely, decreasing mTORC1 activity resulted in relative enrichment of this lowest price furosemide memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also lowest price furosemide found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR.

Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate. Memory B cells and long-lived plasma cells are responsible for effective long-term immunity lowest price furosemide against pathogens. The majority of these cells responding to T cell–dependent antigens are generated from the germinal center (GC) reaction.

Indeed, memory B cells emerge from the GC as recirculating cells and, upon secondary antigen challenge, they are primed to elicit rapid antibody responses lowest price furosemide. GCs are divided into two anatomical structures. The light zone (LZ) and the dark lowest price furosemide zone (DZ.

Allen et al., 2007. Victora and Nussenzweig, lowest price furosemide 2012). B cells proliferate and undergo somatic hypermutation in the DZ before entering the LZ, where they exit the cell cycle.

In the LZ, GC B cells expressing newly mutated B cell receptors (BCRs) capture antigen presented on follicular dendritic cells lowest price furosemide and internalize it for presentation to follicular helper T cells. Subsequently, antigen- and T cell–dependent selection takes place, whereby the “choice” of recycling to the DZ for further affinity maturation or of exiting the GC as plasma or memory B cells is made. In regard to the selection mechanism, it has been lowest price furosemide postulated that precursor cells destined to become recycling GC, plasma, or memory B cells already become committed in the LZ, at least to some extent, thereafter entering the recycling DZ, plasma, or memory B cell pools (Inoue et al., 2018).

For instance, it has been demonstrated that a small fraction of LZ B cells expressing c-Myc, a key cell-cycle regulator, corresponds to precursor cells for the recycling GC fate. C-Myc+ cells are enriched for high-affinity BCRs and ablation of c-Myc lowest price furosemide affects DZ reentry (Calado et al., 2012. Dominguez-Sola et al., 2012.

Finkin et al., 2019) lowest price furosemide. Bcl6loCD69hi LZ B cells expressing IRF4, a critical transcription factor for plasma cell differentiation, were recently shown to be the precursors of plasma cells (Ise et al., 2018). In contrast to these insights into the precursor cells for recycling and plasma cell fates, studies of the memory fate decision have been hampered by the lack of a known master transcription lowest price furosemide factor for differentiation of memory B cells.

Hence, surrogate markers such as an S1PR2 reporter, CCR6 expression, or a cell cycle reporter have been recently employed for identification of memory precursor cells (Laidlaw et al., 2017. Suan et al., lowest price furosemide 2017. Wang et al., 2017).

Although informative, these studies have not identified key features for development of the lowest price furosemide GC-derived precursor cells committed to the long-lived memory B cell fate, or what signals regulate these key features. Here, after identifying a memory-prone population (CD38intBcl6hi/int Ephrin-B1 [Efnb1+]), we found that this small population exhibited lower mTORC1 activity than the recycling-prone population. Constitutive high mTORC1 activity led to defective lowest price furosemide development of CD38intBcl6hi/intEfnb1+ cells, whereas decreasing mTORC1 activity resulted in relative enrichment in this memory-prone cell population versus the recycling-prone one.

Moreover, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR, thereby contributing to their survival and development. We also found that downregulation of lowest price furosemide Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity (Ersching et al., 2017), our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC cells to assume the memory B cell fate.

To clarify the initiating process for memory B cell differentiation occurring in the GC, we wished to identify lowest price furosemide GC B cells destined to the memory fate. For this, we used Bcl6 protein reporter mice (Kitano et al., 2011). We immunized lowest price furosemide these mice with 4-hydroxy-3-nitrophenylacetyl (NP)–chicken γ-globulin (CGG) in alum i.p.

And analyzed NP-specific IgG1+ splenic B cells at day 10. Since CD38 upregulation takes place during the transition from GC to memory B cells (Ridderstad lowest price furosemide and Tarlinton, 1998), we examined such CD38+ B cells that still maintained GC identity to some extent, i.e., were Bcl6+, together with conventional CD38− GC B cells. By using a fractionation method described previously (Fig.

S1 A lowest price furosemide. Ise et al., 2018), the LZ B cells were further separated based on their Bcl6 and CD69 expression pattern (upper right panel in Fig. 1 A) lowest price furosemide.

Fraction (Fr.) 1 (CD38−Bcl6loCD69hi) and Fr.2 (CD38−Bcl6hiCD69hi) cells are lowest price furosemide plasma and recycling GC precursor cells, respectively (Ise et al., 2018). Characterization of Fr.3 (CD38−Bcl6hiCD69lo) cells is described below. Efnb1 is expressed at a high lowest price furosemide level by almost all Fas+GL7+ cells, but is barely detectable on naive B cells (Laidlaw et al., 2017.

Lu et al., 2017. Wang et al., 2017), allowing us lowest price furosemide to identify transitional populations between GC and memory B cells. Hence, for CD38+ cells, by using Efnb1 and Bcl6, we further separated the NP+ IgG1+CD38+GL7−CD138− cells into Bcl6+Efnb1+ (Fr.5), Bcl6loEfnb1+ (Fr.6), and Bcl6−Efnb1− (Fr.7.

Lower right panel in Fig lowest price furosemide. 1 A). Since expression level of Bcl6 in Fr.5 cells was slightly but significantly lower than that of Fr.3 cells, lowest price furosemide as shown by the left panel in Fig.

1 B, herein, we designated Bcl6hi/int for Fr.5. CD38 expression levels on Fr.5, Fr.6, and Fr.7 cells were increased in that order (middle lowest price furosemide panel in Fig. 1 B.

Herein, indicated lowest price furosemide as CD38int, and CD38+ for Fr.5 and 6/7, respectively). During the time course of the GC response, Fr.5 and Fr.6 cell numbers peaked at day 10 before declining, whereas Fr.7 cells peaked at day 12 and then slowly declined (Fig. S1 B) lowest price furosemide.

These kinetic data suggest that Fr.5 and Fr.6 contain cells that are transient and intermediate, and that once cells enter the Fr.7 pool, they are stably maintained. The Fr.7 cells displayed a typical CD38+Bcl6−Efnb1− mature memory phenotype (Fig lowest price furosemide. 1 B).

To assess the relationship between overall LZ B lowest price furosemide cells and Fr.5/6/7 cells, we performed RNA sequencing (RNA-seq) analysis (Fig. S2 A). To obtain sufficient amounts of RNA for this analysis, we used transferred B1-8hi lowest price furosemide B cells instead of non-BCR transgenic mice.

These NP-specific transgenic GC B cells were present in similar proportions in each fraction as in non-BCR transgenic mice (Fig. S1 C) lowest price furosemide. The principal component analysis (PCA) for each fraction indicated that memory B cells (Fr.7) clustered most tightly with CD38+Bcl6loEfnb1+ (Fr.6) cells but differed greatly from total LZ GC B cells (Fig.

1 C) lowest price furosemide. Fr.5 cells were intermediate between Fr.6 and LZ GC B cells. Fr.6 cells expressed lower levels of S1pr2 and higher levels of Gpr183 (EBI2) mRNA compared with LZ B cells lowest price furosemide (Fig.

S1 D), implying that they are a cell population in the process of exiting the GC. Herein, we call Fr.6 “pre-memory B cells.” In contrast to Fr.6 and mature memory B cells (Fr.7), Fr.5 lowest price furosemide cells seem to start the process of downregulating Bcl6. Fr.6 cells are most likely to correspond to the already identified GC-derived pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”.

Laidlaw et al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang et lowest price furosemide al., 2017) in that, like those cells, Fr.6 cells are Bcl6int/loBach2int (Fig. S3, A and B). The above data prompted us to consider that, among Fr.2, Fr.3, and Fr.5 cells, the CD38intBcl6hi/intEfnb1+ cells (Fr.5) lowest price furosemide could be potential GC-derived precursors of the pre-memory B cells (Fr.6).

To test this possibility, we took the following three approaches. First, PCA of the lowest price furosemide RNA-seq data was performed, indicating that CD38intBcl6hi/intEfnb1+ cells (Fr.5) and pre-memory B cells (Fr.6) clustered most closely together (Fig. 1 D).

Second, to monitor cellular quiescence, we employed lowest price furosemide mVenus-p27K− transgenic mice, in which mainly G0 phase cells are labeled (Oki et al., 2014), demonstrating that in contrast to Fr.2 and Fr.3 cells, Fr.5 and Fr.6 cells had more mVenus-p27K− probe–positive, i.e., quiescent cells (Fig. 1 E). Finally, in order to assess the memory recall potential of the Fr.5 cells, we used a previously described adoptive lowest price furosemide transfer method (Wang et al., 2017).

As illustrated in Fig. 1 F, Fr.2, Fr.3, Fr.5, or Fr.6 cells were isolated from NP-CGG/alum immunized mice and adoptively transferred (2 × 104 cells per mouse) into sublethally lowest price furosemide irradiated recipient mice together with CD4+ T cells isolated from CGG-immunized mice. The recipient mice were then challenged lowest price furosemide with NP-CGG and analyzed on day 6 for NP-specific plasma cells.

Although less proficient than pre-memory B cells (Fr.6), the ability of the adoptively transferred CD38intBcl6hi/intEfnb1+ (Fr.5) cells to give rise to plasma cells was significantly superior to Fr.2 and Fr.3 cells (Fig. 1 G) lowest price furosemide. To rule out the possibility that Fr.5 cells were cells that had reentered the GC reaction from already generated memory B cells, we stained them for Ki67 and observed lower expression in Fr.5 than in the pre-GC B cells, which are in the process of entering the GC (Fig.

S1 E) lowest price furosemide. Together, CD38intBcl6hi/intEfnb1+ (Fr.5) cells are likely to be a precursor of pre-memory B cells, herein called Fr.5 “pro-memory B cells,” and to represent a precursor population of previously identified pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”. Laidlaw et lowest price furosemide al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang et al., 2017.

Fig. S3, A and lowest price furosemide B). However, we do not exclude the possibility that the pro-memory B cell population (Fr.5) is heterogeneous in its origins and properties.

For instance, Fr.5 cells appear to overlap, to some extent, with LZ lowest price furosemide CCR6+ cells in that they are beginning to express Ccr6 (Fig. S3 C). To gain insight into the specific features of CD38intBcl6hi/intEfnb1+ (Fr.5) cells that promote their potential development and/or differentiation into memory cells, we compared their RNA-seq lowest price furosemide profile to that of the other LZ B cells (Fr.2 and Fr.3.

Fig. 2 A lowest price furosemide and Fig. S2 A).

CD38−Bcl6hiCD69hi (Fr.2) cells are destined to the recycling GC fate (Ise et al., lowest price furosemide 2018). Gene set enrichment analysis (GSEA) of Hallmark gene sets (Liberzon et al., 2015) revealed a strong enrichment in Fr.2 cells of c-Myc targets, E2F targets, and mTORC1 signaling genes (Fig. S4 A) lowest price furosemide.

Consistent with the mRNA analysis, expression of c-Myc protein, mTORC1 activity (assessed by phospho-S6), and E2F activity (assessed by phospho-Rb) were significantly decreased in Fr.5 cells (Fig. S4 B) lowest price furosemide. In support of this, when we produced anti-NP IgHV186.2 Igλ monoclonal antibodies cloned from single cell-sorted Fr.2 and Fr.5 NP+IgG1+ B cells and measured their relative affinity for NP29- or NP1-BSA, we found a significant overrepresentation of lower-affinity antibodies in CD38intBcl6hi/intEfnb1+ (Fr.5) cells (Fig.

2 B) lowest price furosemide. Consistently, the frequency of canonical affinity–improving mutation (replacement of Trp33 with Leu33. W33L+) was lowest price furosemide lower in Fr.5 cells (Fig.

2 C). Hence, we conclude that, in contrast to CD38−Bcl6hiCD69hi (Fr.2) cells, most of the Fr.5 cells possess lower-affinity BCRs, an indication lowest price furosemide that they received less T cell help in the LZ (Victora et al., 2010). We next compared the RNA-seq profile of Fr.3 to Fr.5 cells (Fig.

2 A lowest price furosemide and Fig. S2 A). Some differences were observed between these two lowest price furosemide fractions.

Particularly, expression of some of mTORC1 signaling genes was higher in Fr.3 than Fr.5 cells (Fig. 2 D) lowest price furosemide. Myc expression in Fr.3 cells was somewhat higher compared with Fr.5 cells (Fig.

2 D) lowest price furosemide. Reflecting these differences, GSEA showed an enrichment in Fr.3 of c-Myc targets and mTORC1 signaling genes (Fig. 2 E), although the enrichment extent of Fr.3 to Fr.5 was much smaller than Fr.2 to Fr.5 cells (Fig lowest price furosemide.

S4 C). By flow cytometry analysis lowest price furosemide of c-Myc and pS6, however, we could not detect significant differences in both c-Myc protein expression and mTORC1 activity between Fr.3 and Fr.5 cells (Fig. S5 A).

These data suggest that our flow cytometry analysis might lowest price furosemide not have sufficed to detect small changes induced by differential mRNA levels between Fr.3 and Fr.5 cells. An alternative possibility is that, lowest price furosemide in addition to mRNA level, changes in translational/posttranslational regulation might take place between Fr.3 and Fr.5 cells. The potential reason why Fr.5 but not Fr.3 cells can become pro-memory B cells, despite relatively small differences in RNA-seq profiles between these two populations, is described below.

To identify lowest price furosemide key properties for the development of Fr.5 cells and/or their activity, we considered that Bach2/Blimp1 double-deficient GC B cells could provide a clue, since these mutant cells are defective in generating GC-derived memory B cells (Shinnakasu et al., 2016). To this end, we transferred B cells of three genotypes (Bach2f/fPrdm1f/fERT2cre B1-8hi, Bach2+/+Prdm1f/fERT2cre B1-8hi, and Bach2+/+Prdm1+/+ERT2cre B1-8hi) into recipient mice, treated them with tamoxifen, and then immunized them with NP-CGG/alum (Fig. 3 A) lowest price furosemide.

In contrast to the control wild-type and Blimp1 single-deficient B cells, Bach2/Blimp1 double-deficient GC B cells showed an enrichment in DZ cells (Fig. 3 B) lowest price furosemide. Moreover, the relatively small proportion of LZ B cells still contained Fr.2 and Fr.3 cells, whereas the numbers of Fr.5 and Fr.7 cells were robustly decreased in Bach2/Blimp1 double-deficient B cells (Fig.

3 B) lowest price furosemide. Since Blimp1 single knockout did not significantly affect the numbers of pro-memory (Fr.5) and mature memory B cells (Fr.7. Fig.

3 B), we conclude that Bach2 plays an important role in development of pro-memory cells and subsequent mature memory B cells. To determine how Bach2 participates in this process, we performed RNA profiling of Bach2/Blimp1 double-deficient LZ B cells, together with Blimp1-deficient LZ B cells as a control (Fig. S2 B).

In Bach2/Blimp1 double-deficient LZ B cells, GSEA revealed a significant enrichment of c-Myc target genes, E2F target genes, and mTORC1 signaling genes, in that order (Fig. 3 C). This was also demonstrated by flow cytometry analysis (expression levels of c-Myc, pRb, and pS6.

Fig. 3 D). Moreover, as expected, the mutant GC B cells were hyperproliferative, as assessed by 5-ethynyl-2′-deoxyuridine (EdU) pulse labeling (Fig.

3 E). These results, considering the previous demonstration that c-Myc–overexpressing and hyper-mTORC1 GC B cells manifest a bias toward the DZ (Ersching et al., 2017. Finkin et al., 2019), like Bach2/Blimp1 double-deficient GC B cells, allowed us to hypothesize that the defective pro-memory in the mutant GC cells 5could result from anomalies of the mTORC1 and/or c-Myc pathways.

Here, we focused our analysis on the mTORC1 pathway. To test this hypothesis, we first asked whether normalizing mTORC1 activity in Bach2/Blimp1 double-deficient GC cells could rescue development of pro-memory B cells and subsequent memory B cells. We transferred Bach2f/fPrdm1f/fERT2cre B1-8hi B cells into rapamycin-resistant (MtorF2108L/F2108L) hosts (Ersching et al., 2017), deleted Bach2 and Prdm1 with tamoxifen, and then immunized the mice with NP-CGG/alum (Fig.

4 A). After immunization, the mice were treated with rapamycin to decrease mTORC1 activity in a transferred B cell–intrinsic manner. As shown in Fig.

4 B, the dose of rapamycin used nearly normalized pS6 levels in the Bach2/Blimp1 double-deficient LZ B cells. The rapamycin treatment partially corrected the c-Myc overexpression and hyperproliferation observed in the Bach2/Blimp1 double-deficient B1-8hi B cells (Fig. 4 B), suggesting coexistence of mTORC1-dependent and -independent pathways to regulate c-Myc activities.

In contrast to control vehicle treatment of Bach2/Blimp1 double-deficient B1-8hi B cells, upon rapamycin treatment, those mutant cells generated threefold higher numbers of IgG1+ memory B cells. The numbers of IgG1+CD73+ memory B cells were similarly increased (Fig. 4 C, right).

Furthermore, the Fr.5:Fr.2 ratio was also increased upon rapamycin treatment (Fig. 4 D). However, the memory B cells number upon rapamycin treatment did not reach those from wild-type B1-8hi B cells upon control vehicle injection (Fig.

4 C). Hence, we conclude that hyper-mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells is one of the mechanisms that cause defective development of memory B cells, although there must be other, currently unknown ones, as well. In regard to GC B cells, the numbers were not significantly changed upon rapamycin treatment of Bach2/Blimp1 double-deficient B1-8hi B cells.

Skewing of Bach2/Blimp1 double-deficient GC B cells toward the DZ was decreased upon rapamycin treatment, although a small enrichment was still observed (Fig. 4 C). To further examine whether, in a wild-type setting, restraining mTORC1 activity could indeed facilitate differentiation of GC B cells to memory cells, we performed adoptive transfer experiments.

For this, we conducted experiments in which two types of congenically marked B cells, rapamycin-sensitive (Mtor+/+) and rapamycin-resistant (MtorF2108L/F2108L) B1-8ge B cells, were cotransferred as a 1:1 mixture into rapamycin-resistant hosts (MtorF2108L/F2108L), which were immunized with NP-CGG/alum and then administered with rapamycin. As expected, rapamycin treatment led to a decrease in S6 phosphorylation in the transferred rapamycin-sensitive, but not rapamycin-resistant, B1-8ge GC B cells (Fig. 5 A).

Upon rapamycin treatment, the number of rapamycin-sensitive NP+ GC B cells was decreased while the number of NP+ memory B cells was increased compared with their rapamycin-resistant counterparts, assessed by conventional flow cytometry analysis (Fig. 5 B). To more directly demonstrate the transition from GC B cells to Fr.7 cells, we treated the immunized mice with EdU for 3 d (days 10–13) before analysis.

In this setting, incorporation of EdU marks GC cells that divided during the treatment period and the resultant quiescent memory B cells (Fig. 5 C). We previously confirmed that during this period, the majority of proliferating cells (>95%) are GC B cells and plasmablasts (Shinnakasu et al., 2016).

Upon rapamycin treatment, the frequency of EdU+IgG1+ Fr.7 cells compared with GC cells was higher among the rapamycin-sensitive B1-8ge cells than the rapamycin-resistant ones, demonstrating rapamycin-mediated facilitation of the transition from GC to Fr.7 cells (Fig. 5 D). Moreover, upon rapamycin treatment, the numbers of CD38−Bcl6hiCD69hi (Fr.2) and CD38intBcl6hi/intEfnb1+ (Fr.5) rapamycin-sensitive B1-8ge IgG1+ B cells were decreased and maintained, respectively.

Thus, the ratio of Fr.5 to Fr.2 was increased (Fig. 5 E). Together, we conclude that a relative enrichment in Fr.5 over Fr.2 cells is induced by rapamycin treatment, thereby facilitating the overall transition from GC B cells to memory B cells.

The memory B cells generated in the presence of rapamycin were able to induce similar recall antibody responses to those generated in the absence of rapamycin, as assessed by adoptive transfer experiments (Fig. 5 F). We next wished to examine why Fr.5, but not Fr.3 cells, can become pro-memory B cells.

Since there were almost no differences in mTORC1 activity between Fr.5 and Fr.3 cells (Fig. S5 A), it appears that an mTORC1lo state is necessary but not sufficient for development of pro-memory B cells (Fr.5). Thus, additional key properties must be required for development of these cells.

Since one of crucial features of mature memory B cells is longevity, one straightforward possibility is that Fr.5 cells begin to acquire more survival activity. Supporting this idea, CD38intBcl6hi/intEfnb1+ (Fr.5) cells were less apoptotic compared with CD38−Bcl6hiCD69lo (Fr.3) cells as assessed by active caspase-3 staining (Fig. 6 A).

Transcript data (Fig. 6 B) together with protein expression data (Fig. 6 C) demonstrated that Bcl2 expression was upregulated in Fr.5 cells compared with Fr.3 cells, and even more in pre-memory B cells (Fr.6).

Similarly, we found that the cell surface BCR expression level was increased stepwise from Fr.3 to Fr.6 cells (Fig. 6 D). We also observed a slight increase of IgG1 and Igα/β mRNA expression in Fr.5 over Fr.3 cells.

Thus, regulation of both mRNA and protein levels seems to be operative. To examine whether Bcl2 family protein–mediated survival activity could impact the development of Fr.5 cells, we employed GC B cells with haploinsufficiency of Bim (Bcl2l11. See Materials and methods), a counteracting factor against anti-apoptotic Bcl2-family members (O’Connor et al., 1998).

Bcl2l11+/+ ERT2cre B1-8ge B cells and Bcl2l11f/+ERT2cre B1-8ge B cells were cotransferred as a 1:1 mixture into wild-type recipient mice, which were then immunized with NP-CGG/alum and treated with tamoxifen on day 8 (Fig. 6 E). Bim mRNA expression was decreased to almost 50% of control levels after tamoxifen treatment in Bcl2l11f/+ GC B cells (Fig.

6 F). In this competitive setting, among the Fr.2/3/5/6 cells, the frequency was most significantly increased in Fr.5 and Fr.6 cells upon Bim haploinsufficiency (Fig. 6 G), although there was also a modest increase of Fr.3 cells.

Consequently, the frequency of Bcl2l11f/+ NP+IgG1+CD73+ memory B cells was also increased (Fig. S5 B). To examine the effects of surface BCR expression on survival, B1-8ge-flox/+ ERT2cre B cells were employed.

For these particular experiments, we mixed these B cells and control B1-8ge/+ ERT2cre B cells at a 7:3 ratio and adoptively cotransferred them into recipient mice, which were then immunized with NP-CGG/alum (Fig. 6 H). We injected tamoxifen on day 10 and examined surface BCR expression on day 12, demonstrating a significant decrease on Fr.5 cells derived from B1-8ge-flox/+ ERT2cre B cells (Fig.

6 I). To detect apoptotic cells in this experiment, we analyzed mixtures of Fr.5 and Fr.6 cells (CD38+Efnb1+) to acquire a sufficient number of cells for the assay. As demonstrated in Fig.

6 J, concomitant with decreased surface BCR expression, there was a higher frequency of apoptotic (aCasp3+) cells among pro/pre-memory cells derived from B1-8ge-flox/+ ERT2cre B cells. Similarly, frequency of apoptotic cells among total LZ GC cells was enhanced upon BCR downregulation (Fig. S5 C).

A control experiment using Prdm1f/+B1-8ge/+ ERT2cre B cells showed that a nonspecific effect on apoptosis induced simply by Cre-mediated double-strand breaks was negligible (Fig. S5 D). Together, stepwise increases of Bcl2 and surface BCR expression from pro-memory (Fr.5) cells to pre-memory (Fr.6) toward mature memory B cells are likely to contribute to their survival.

It is still unclear what signals and processes in LZ GC cells initiate their differentiation toward long-lived memory B cells. Here, by focusing on key features for development of GC-derived memory precursors, we show that an mTORC1lo state is necessary to develop pro-memory B cells. Since mTORC1lo LZ B cells receive weak T cell help and, as a result, have been thought to undergo apoptosis, this raises the question of how such pro-memory B cells are prevented from dying and able to differentiate into mature memory B cells.

Our experiments suggest that the memory precursor B cells express higher levels of Bcl2 and surface BCR, thereby acquiring a survival advantage. We have already shown that Bach2hi LZ GC B cells are predisposed to differentiate into memory B cells (Shinnakasu et al., 2016), indicating that memory cell commitment already begins in a subset of GC B cells. This memory-prone subset most likely corresponds to the Fr.5 (CD38intBcl6hi/intEfnb1+ pro-memory) cells.

Indeed, expression of Bach2 in Fr.5 is higher than in Fr.2 cells (Fig. 2 A and Fig. S3 B).

Fr.6 (pre-memory B) cells appear to be undergoing a further developmental step toward mature memory B cells, manifested by further downregulation of Bcl6 (Fig. 1 B). We found that mTORC1 has a marked effect on the ratio of memory-prone (Fr.5) to recycling-prone (Fr.2) GC B cell formation.

Rapamycin treatment increased the proportion of Fr.5 cells and, conversely, hyperactivation of mTORC1 in the Bach2/Blimp1 double-deficient setting led to a relative increase in Fr.2 cells. Several nonmutually exclusive possibilities can be envisaged to explain why lower mTORC1 activity contributes to development of memory-prone cells. Decay in mTORC1 activity as GC B cells proliferate in the DZ appears to be required for their timely return to the LZ (Ersching et al., 2017).

Given the importance of LZ residency for memory differentiation (Bannard et al., 2013), one possibility is that LZ residency imposed by mTORC1lo could allow development of pro-memory B cells. Second, apart from this spatial requirement mediated through modulation of mTORC1, inhibition of mTORC1, as is seen during the generation of natural killer cell memory (O’Sullivan et al., 2015), may stimulate autophagy, thereby enhancing pro-memory B cell survival. Finally, it is also well known that mTORC1 activity is suppressed in memory B cells (Boothby and Rickert, 2017).

Such metabolic changes as the cells progress toward mature memory B cells thus appear to be initiated already in pro-memory cells, and this might be a necessary first step for generating mature memory B cells. The partial restoration of memory B cells by rapamycin treatment in Bach2/Blimp1 double-deficient GC cells suggests that, in addition to hyper-mTORC1 activity, other anomalies occur in mutant GC B cells in regard to memory differentiation. One of them is likely the c-Myc overexpression, because of the following.

First, indeed, in rapamycin-treated Bach2/Blimp1 double-deficient GC cells, overexpression of c-Myc and hyperproliferation were still observed to a significant extent (Fig. 4 B). Second, c-Myc–overexpressing GC cells were reported to have a significant bias toward the DZ (Finkin et al., 2019).

Considering the importance of LZ residency for memory differentiation (Bannard et al., 2013), overexpression of c-Myc is assumed to be detrimental to memory differentiation. Hence, we would propose that restraining both mTORC1-mediated metabolism and c-Myc–mediated cell-cycle progression is required to develop pro-memory B cells and that Bach2 is one of the critical regulators for suppressing both pathways. Functionally, Bach2 is well known to act as a repressive guardian transcription factor (Igarashi et al., 2017).

In regard to relationship between signaling and Bach2 expression, the mTORC1 activity and Bach2 expression appear to be mutually exclusive, because the BCR-induced AKT-mTORC1 inhibits Bach2 expression (Kometani et al., 2013), and Bach2 represses transcription of mTORC1 signaling molecules. Such a negative feedback loop is characteristic of “bistable” signal transduction circuits, which can operate in two stable formats. This might take place between Fr.5 and Fr.2 cells.

It should be mentioned that, from mTORC1 signaling molecule side, Bach2 is one of the transcription factors, and probably additional factors participate in transcriptional regulation on mTORC1 signaling genes. In addition to the connection between BCR signal and Bach2, considering the T cell data showing that ICOS and integrin αE are upregulated in Bach2lo T cells (Grant et al., 2020. Sidwell et al., 2020), Bach2 might be involved in connecting the BCR signal to T cell help.

For instance, Bach2lo LZ GC cells with high-affinity BCRs might modulate T/B interactions through adhesion status and coreceptor expression and affect the strength of T cell help. This might further downregulate Bach2, because we previously showed that strong T cell help depresses Bach2 expression (Shinnakasu et al., 2016). After moving back into the LZ, apoptosis is generally thought to be the default pathway for LZ GC B cells.

However, high-affinity cells are spared and positively selected after they encounter sufficient cognate T cell help (Allen et al., 2007. Victora and Nussenzweig, 2012). These spared high-affinity cells correspond to Fr.2 cells, whereas the defaulting apoptotic LZ cells are likely to be Fr.3 cells.

Indeed, among LZ GC cells, Fr.3 cells were most apoptotic. Here, we show that a small population of pro-memory B cells exists in the LZ and, despite apparently receiving weak T cell help, they are relatively resistant to apoptosis. The inability of prior studies to detect such apoptosis-resistant LZ B cells is most likely due to the fact that the numbers of pro-memory cells are so limited (Mayer et al., 2017).

Previous data using B cell–specific Bcl2-tg mice (Smith et al., 1994) or Bim knockout mice (Fischer et al., 2007) showed that such mice develop an enlarged memory B cell compartment. Recently, more detailed analysis using the same Bcl2-tg mice (Stewart et al., 2018) provided mechanistic insights into the above phenomenon. First, in these mice, aberrant populations of seemingly quiescent cells arise that express markers of memory precursor cells.

Second, overexpression of Bcl2 is not sufficient for DZ GC B cells with damaged BCRs to reach the LZ. Hence, in a physiological setting, it is reasonable to speculate that, after returning to the LZ in a Bcl2-independent manner, if Bcl2 expression is upregulated in some of the LZ GC B cells, they are better able to be rescued from apoptosis in the late G1 phase and to begin to differentiate into memory B cells. Supporting this idea, we show here that among LZ GC cells, small numbers of pro-memory B cells (Fr.5), but not Fr.3 cells, begin to upregulate Bcl2, and that development of pro-memory B cells is facilitated by Bim haploinsufficiency.

Because Bach2 expression in Fr.5 cells is similar to Fr.3 cells (Fig. S3 B), Bach2 appears not to be involved in such differential survival activity between Fr.5 and Fr.3 cells. Rather, a Bach2-independent mechanism such as Bcl6 downregulation (discussed below) is likely to be operated, thereby allowing Fr.5 cells to survive enough to begin to differentiate into memory precursor cells.

In contrast to Fr.5 cells (pro-memory), Fr.6 cells (pre-memory) apparently possess more survival activity (Fig. 6 A), possibly explaining the generation kinetics between Fr.5 and Fr.6 cells. Fr.6 cells were more accumulated at later phases (days 14 and 20) during immune responses (Fig.

S1 B). Induced downregulation of surface BCR expression in pro/pre-memory B cells resulted in increased apoptosis in the pro-memory B cells. These results, together with the evidence that pro-memory B cells express higher surface BCR levels, lead us to propose that the BCR-mediated survival signal also plays a role in the development of pro-memory B cells.

Based on the previous report that BCR ablation leads to cell death, which can be delayed by constitutive Bcl2 expression (Lam et al., 1997), we considered the possibility that downregulation of the BCR might decrease Bcl2 expression in pro/pre-memory B cells. However, we could not detect such a connection (data not shown). In naive B cells, the constitutive PI3 kinase–Foxo1 pathway is known to replace the missing BCR-mediated survival signals (Srinivasan et al., 2009).

Therefore, a question arises of how pro-memory B cells, despite being mTORC1lo (reflecting lower Akt activity), generate such a survival signal. Given that there is no enlarged GC phenotype in PTEN or Foxo1 knockout mice (Dominguez-Sola et al., 2015. Inoue et al., 2017.

Sander et al., 2015. Suzuki et al., 2003), one straightforward explanation might be that the quality and/or quantity of BCR-mediated survival signals differ between naive B cells and GC-derived memory B cells. We provide evidence that downregulation of Bcl6 in pro-memory B cells could be one of the mechanisms for upregulation of Bcl2 and surface BCR.

However, since the extent of Bcl6 downregulation in pro-memory B cells is small, such a slight change might not account for the observed upregulation of Bcl2 and surface BCR. Hence, our data cannot completely exclude the possibility that, particularly at the pro-memory B cell stage, other mechanisms might operate to initiate upregulation of Bcl2 and BCR. In this case, it is likely that downregulation of Bcl6 acts as an amplification pathway for further upregulation of Bcl2 and surface BCR during differentiation toward mature memory B cells.

In regard to this differentiation pathway, our data using Bcl6 haploinsufficiency are highly complementary to previous in vitro data that ectopic expression of Bcl6 in B cell cultures blocks the GC B cells from differentiating into memory B cells (Kuo et al., 2007). Together, it is likely that stepwise decreases in Bcl6 expression (pro-memory >. Pre-memory >.

Mature memory B cells) play a key role in memory B cell development. This raises the question of how is Bcl6 downregulated. Three possibilities have already been reported.

(1) upon strong BCR engagement, Erk-mediated degradation of Bcl6 (Niu et al., 1998). (2) transcriptional downregulation of Bcl6, mediated by CD40-activated IRF4 (Saito et al., 2007). And (3) downregulation of Bcl6 by defective IL-21 signaling (Linterman et al., 2010).

Among these, in regard to differentiation from GC to memory B cells, the final possibility seems to best fit with our observation that pro-memory B cells possess lower-affinity BCRs, thereby receiving less T cell help. In addition, as a transcriptional circuit–type regulation, the transcription factor Hhex, critical for memory B cell differentiation, has been recently reported to participate in downregulation of Bcl6 (Laidlaw et al., 2020). In summary, this study provides important insights into the initial events for the fate decisions from GC to memory B cells.

The modulation of cellular metabolism and survival play fundamental roles. Given the importance of GC-derived memory B cells for protection against heterologous virus reinfection (Leach et al., 2019. Purtha et al., 2011), our findings may contribute to the development of efficient vaccination strategies.

Single-cell suspensions of splenocytes were analyzed and sorted on a FACSCanto II (BD Biosciences) or a FACSAria II (BD Biosciences). Alexa647-active caspase-3, V500-B220, V450-Bcl6, BV786-CD138, BV510-CD38, V500-CD45.2, BV510-IgG1, PE-IgG1 antibodies, and BV786-streptavidin were purchased from BD Biosciences. APC-eFluor780-B220, FITC-CD45.1, PE-CD45.1, APC-eFluor780-CD45.1, FITC-CD45.2, APC-eFluor780-CD45.2, APC-CD69, APC-eFluor780-CD69, eFluor450-CD73, PE-CD86, PerCP-Cy5.5-GL7 antibodies, PerCP-Cy5.5-streptavidin, PE-streptavidin, and APC-eFluor780-streptavidin were purchased from eBioscience.

PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. C-Myc antibody was purchased from Abcam.

PE-Bcl2 antibody was purchased from Miltenyi Biotec. Biotin-Efnb1 antibody was purchased from R&D Systems. Alexa488-goat anti-rabbit IgG antibody was purchased from Thermo Fisher Scientific.

For intracellular staining, the cells were fixed and permeabilized using a Foxp3 staining kit (eBioscience) for Bcl6, Bcl2, and pRb, a BD Cytofix/Cytoperm solution (BD Biosciences) for pS6 and active caspase-3, or a True-Nuclear Transcription Factor Staining Buffer Set (BioLegend) for c-Myc. APC-conjugated NP was prepared as described previously (Shinnakasu et al., 2016). Incorporation of EdU was detected using a Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

We thank M.C. Nussenzweig (The Rockefeller University, New York, NY) for B1-8hi mice, T. Okada (RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan) for Bcl6-YFP mice, G.D.

Victora (The Rockefeller University) for MtorF2108L mice, and P.D. Burrows for critical reading of the manuscript. This work was supported by grants from JSPS KAKENHI (JP17K08882 to T.

Inoue. JP26221306 and JP19H01028 to T. Kurosaki), the SENSHIN Medical Research Foundation (to T.

Inoue), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. Inoue), and a research grant from Astellas Foundation for Research on Metabolic Disorders (to T. Inoue).

Kawai performed the experiments. T. Inoue and T.

Kurosaki designed the experiments. R. Shinnakasu, W.

Fukuyama provided essential reagents. E. Kawakami, N.

Sax, and K. Yamashita performed bioinformatics analyses. T.

Inoue and T. Kurosaki wrote the manuscript.A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate.

Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells. Conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development.

We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate. Memory B cells and long-lived plasma cells are responsible for effective long-term immunity against pathogens.

The majority of these cells responding to T cell–dependent antigens are generated from the germinal center (GC) reaction. Indeed, memory B cells emerge from the GC as recirculating cells and, upon secondary antigen challenge, they are primed to elicit rapid antibody responses. GCs are divided into two anatomical structures.

The light zone (LZ) and the dark zone (DZ. Allen et al., 2007. Victora and Nussenzweig, 2012).

B cells proliferate and undergo somatic hypermutation in the DZ before entering the LZ, where they exit the cell cycle. In the LZ, GC B cells expressing newly mutated B cell receptors (BCRs) capture antigen presented on follicular dendritic cells and internalize it for presentation to follicular helper T cells. Subsequently, antigen- and T cell–dependent selection takes place, whereby the “choice” of recycling to the DZ for further affinity maturation or of exiting the GC as plasma or memory B cells is made.

In regard to the selection mechanism, it has been postulated that precursor cells destined to become recycling GC, plasma, or memory B cells already become committed in the LZ, at least to some extent, thereafter entering the recycling DZ, plasma, or memory B cell pools (Inoue et al., 2018). For instance, it has been demonstrated that a small fraction of LZ B cells expressing c-Myc, a key cell-cycle regulator, corresponds to precursor cells for the recycling GC fate. C-Myc+ cells are enriched for high-affinity BCRs and ablation of c-Myc affects DZ reentry (Calado et al., 2012.

Dominguez-Sola et al., 2012. Finkin et al., 2019). Bcl6loCD69hi LZ B cells expressing IRF4, a critical transcription factor for plasma cell differentiation, were recently shown to be the precursors of plasma cells (Ise et al., 2018).

In contrast to these insights into the precursor cells for recycling and plasma cell fates, studies of the memory fate decision have been hampered by the lack of a known master transcription factor for differentiation of memory B cells. Hence, surrogate markers such as an S1PR2 reporter, CCR6 expression, or a cell cycle reporter have been recently employed for identification of memory precursor cells (Laidlaw et al., 2017. Suan et al., 2017.

Wang et al., 2017). Although informative, these studies have not identified key features for development of the GC-derived precursor cells committed to the long-lived memory B cell fate, or what signals regulate these key features. Here, after identifying a memory-prone population (CD38intBcl6hi/int Ephrin-B1 [Efnb1+]), we found that this small population exhibited lower mTORC1 activity than the recycling-prone population.

Constitutive high mTORC1 activity led to defective development of CD38intBcl6hi/intEfnb1+ cells, whereas decreasing mTORC1 activity resulted in relative enrichment in this memory-prone cell population versus the recycling-prone one. Moreover, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR, thereby contributing to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR.

Given the positive correlation between the strength of T cell help and mTORC1 activity (Ersching et al., 2017), our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC cells to assume the memory B cell fate. To clarify the initiating process for memory B cell differentiation occurring in the GC, we wished to identify GC B cells destined to the memory fate. For this, we used Bcl6 protein reporter mice (Kitano et al., 2011).

We immunized these mice with 4-hydroxy-3-nitrophenylacetyl (NP)–chicken γ-globulin (CGG) in alum i.p. And analyzed NP-specific IgG1+ splenic B cells at day 10. Since CD38 upregulation takes place during the transition from GC to memory B cells (Ridderstad and Tarlinton, 1998), we examined such CD38+ B cells that still maintained GC identity to some extent, i.e., were Bcl6+, together with conventional CD38− GC B cells.

By using a fractionation method described previously (Fig. S1 A. Ise et al., 2018), the LZ B cells were further separated based on their Bcl6 and CD69 expression pattern (upper right panel in Fig.

1 A). Fraction (Fr.) 1 (CD38−Bcl6loCD69hi) and Fr.2 (CD38−Bcl6hiCD69hi) cells are plasma and recycling GC precursor cells, respectively (Ise et al., 2018). Characterization of Fr.3 (CD38−Bcl6hiCD69lo) cells is described below.

Efnb1 is expressed at a high level by almost all Fas+GL7+ cells, but is barely detectable on naive B cells (Laidlaw et al., 2017. Lu et al., 2017. Wang et al., 2017), allowing us to identify transitional populations between GC and memory B cells.

Hence, for CD38+ cells, by using Efnb1 and Bcl6, we further separated the NP+ IgG1+CD38+GL7−CD138− cells into Bcl6+Efnb1+ (Fr.5), Bcl6loEfnb1+ (Fr.6), and Bcl6−Efnb1− (Fr.7. Lower right panel in Fig. 1 A).

Since expression level of Bcl6 in Fr.5 cells was slightly but significantly lower than that of Fr.3 cells, as shown by the left panel in Fig. 1 B, herein, we designated Bcl6hi/int for Fr.5. CD38 expression levels on Fr.5, Fr.6, and Fr.7 cells were increased in that order (middle panel in Fig.

1 B. Herein, indicated as CD38int, and CD38+ for Fr.5 and 6/7, respectively). During the time course of the GC response, Fr.5 and Fr.6 cell numbers peaked at day 10 before declining, whereas Fr.7 cells peaked at day 12 and then slowly declined (Fig.

S1 B). These kinetic data suggest that Fr.5 and Fr.6 contain cells that are transient and intermediate, and that once cells enter the Fr.7 pool, they are stably maintained. The Fr.7 cells displayed a typical CD38+Bcl6−Efnb1− mature memory phenotype (Fig.

1 B). To assess the relationship between overall LZ B cells and Fr.5/6/7 cells, we performed RNA sequencing (RNA-seq) analysis (Fig. S2 A).

To obtain sufficient amounts of RNA for this analysis, we used transferred B1-8hi B cells instead of non-BCR transgenic mice. These NP-specific transgenic GC B cells were present in similar proportions in each fraction as in non-BCR transgenic mice (Fig. S1 C).

The principal component analysis (PCA) for each fraction indicated that memory B cells (Fr.7) clustered most tightly with CD38+Bcl6loEfnb1+ (Fr.6) cells but differed greatly from total LZ GC B cells (Fig. 1 C). Fr.5 cells were intermediate between Fr.6 and LZ GC B cells.

Fr.6 cells expressed lower levels of S1pr2 and higher levels of Gpr183 (EBI2) mRNA compared with LZ B cells (Fig. S1 D), implying that they are a cell population in the process of exiting the GC. Herein, we call Fr.6 “pre-memory B cells.” In contrast to Fr.6 and mature memory B cells (Fr.7), Fr.5 cells seem to start the process of downregulating Bcl6.

Fr.6 cells are most likely to correspond to the already identified GC-derived pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”. Laidlaw et al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang et al., 2017) in that, like those cells, Fr.6 cells are Bcl6int/loBach2int (Fig. S3, A and B).

The above data prompted us to consider that, among Fr.2, Fr.3, and Fr.5 cells, the CD38intBcl6hi/intEfnb1+ cells (Fr.5) could be potential GC-derived precursors of the pre-memory B cells (Fr.6). To test this possibility, we took the following three approaches. First, PCA of the RNA-seq data was performed, indicating that CD38intBcl6hi/intEfnb1+ cells (Fr.5) and pre-memory B cells (Fr.6) clustered most closely together (Fig.

1 D). Second, to monitor cellular quiescence, we employed mVenus-p27K− transgenic mice, in which mainly G0 phase cells are labeled (Oki et al., 2014), demonstrating that in contrast to Fr.2 and Fr.3 cells, Fr.5 and Fr.6 cells had more mVenus-p27K− probe–positive, i.e., quiescent cells (Fig. 1 E).

Finally, in order to assess the memory recall potential of the Fr.5 cells, we used a previously described adoptive transfer method (Wang et al., 2017). As illustrated in Fig. 1 F, Fr.2, Fr.3, Fr.5, or Fr.6 cells were isolated from NP-CGG/alum immunized mice and adoptively transferred (2 × 104 cells per mouse) into sublethally irradiated recipient mice together with CD4+ T cells isolated from CGG-immunized mice.

The recipient mice were then challenged with NP-CGG and analyzed on day 6 for NP-specific plasma cells. Although less proficient than pre-memory B cells (Fr.6), the ability of the adoptively transferred CD38intBcl6hi/intEfnb1+ (Fr.5) cells to give rise to plasma cells was significantly superior to Fr.2 and Fr.3 cells (Fig. 1 G).

To rule out the possibility that Fr.5 cells were cells that had reentered the GC reaction from already generated memory B cells, we stained them for Ki67 and observed lower expression in Fr.5 than in the pre-GC B cells, which are in the process of entering the GC (Fig. S1 E). Together, CD38intBcl6hi/intEfnb1+ (Fr.5) cells are likely to be a precursor of pre-memory B cells, herein called Fr.5 “pro-memory B cells,” and to represent a precursor population of previously identified pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”.

Laidlaw et al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang et al., 2017. Fig. S3, A and B).

However, we do not exclude the possibility that the pro-memory B cell population (Fr.5) is heterogeneous in its origins and properties. For instance, Fr.5 cells appear to overlap, to some extent, with LZ CCR6+ cells in that they are beginning to express Ccr6 (Fig. S3 C).

To gain insight into the specific features of CD38intBcl6hi/intEfnb1+ (Fr.5) cells that promote their potential development and/or differentiation into memory cells, we compared their RNA-seq profile to that of the other LZ B cells (Fr.2 and Fr.3. Fig. 2 A and Fig.

S2 A). CD38−Bcl6hiCD69hi (Fr.2) cells are destined to the recycling GC fate (Ise et al., 2018). Gene set enrichment analysis (GSEA) of Hallmark gene sets (Liberzon et al., 2015) revealed a strong enrichment in Fr.2 cells of c-Myc targets, E2F targets, and mTORC1 signaling genes (Fig.

S4 A). Consistent with the mRNA analysis, expression of c-Myc protein, mTORC1 activity (assessed by phospho-S6), and E2F activity (assessed by phospho-Rb) were significantly decreased in Fr.5 cells (Fig. S4 B).

In support of this, when we produced anti-NP IgHV186.2 Igλ monoclonal antibodies cloned from single cell-sorted Fr.2 and Fr.5 NP+IgG1+ B cells and measured their relative affinity for NP29- or NP1-BSA, we found a significant overrepresentation of lower-affinity antibodies in CD38intBcl6hi/intEfnb1+ (Fr.5) cells (Fig. 2 B). Consistently, the frequency of canonical affinity–improving mutation (replacement of Trp33 with Leu33.

W33L+) was lower in Fr.5 cells (Fig. 2 C). Hence, we conclude that, in contrast to CD38−Bcl6hiCD69hi (Fr.2) cells, most of the Fr.5 cells possess lower-affinity BCRs, an indication that they received less T cell help in the LZ (Victora et al., 2010).

We next compared the RNA-seq profile of Fr.3 to Fr.5 cells (Fig. 2 A and Fig. S2 A).

Some differences were observed between these two fractions. Particularly, expression of some of mTORC1 signaling genes was higher in Fr.3 than Fr.5 cells (Fig. 2 D).

Myc expression in Fr.3 cells was somewhat higher compared with Fr.5 cells (Fig. 2 D). Reflecting these differences, GSEA showed an enrichment in Fr.3 of c-Myc targets and mTORC1 signaling genes (Fig.

2 E), although the enrichment extent of Fr.3 to Fr.5 was much smaller than Fr.2 to Fr.5 cells (Fig. S4 C). By flow cytometry analysis of c-Myc and pS6, however, we could not detect significant differences in both c-Myc protein expression and mTORC1 activity between Fr.3 and Fr.5 cells (Fig.

S5 A). These data suggest that our flow cytometry analysis might not have sufficed to detect small changes induced by differential mRNA levels between Fr.3 and Fr.5 cells. An alternative possibility is that, in addition to mRNA level, changes in translational/posttranslational regulation might take place between Fr.3 and Fr.5 cells.

The potential reason why Fr.5 but not Fr.3 cells can become pro-memory B cells, despite relatively small differences in RNA-seq profiles between these two populations, is described below. To identify key properties for the development of Fr.5 cells and/or their activity, we considered that Bach2/Blimp1 double-deficient GC B cells could provide a clue, since these mutant cells are defective in generating GC-derived memory B cells (Shinnakasu et al., 2016). To this end, we transferred B cells of three genotypes (Bach2f/fPrdm1f/fERT2cre B1-8hi, Bach2+/+Prdm1f/fERT2cre B1-8hi, and Bach2+/+Prdm1+/+ERT2cre B1-8hi) into recipient mice, treated them with tamoxifen, and then immunized them with NP-CGG/alum (Fig.

3 A). In contrast to the control wild-type and Blimp1 single-deficient B cells, Bach2/Blimp1 double-deficient GC B cells showed an enrichment in DZ cells (Fig. 3 B).

Moreover, the relatively small proportion of LZ B cells still contained Fr.2 and Fr.3 cells, whereas the numbers of Fr.5 and Fr.7 cells were robustly decreased in Bach2/Blimp1 double-deficient B cells (Fig. 3 B). Since Blimp1 single knockout did not significantly affect the numbers of pro-memory (Fr.5) and mature memory B cells (Fr.7.

Fig. 3 B), we conclude that Bach2 plays an important role in development of pro-memory cells and subsequent mature memory B cells. To determine how Bach2 participates in this process, we performed RNA profiling of Bach2/Blimp1 double-deficient LZ B cells, together with Blimp1-deficient LZ B cells as a control (Fig.

S2 B). In Bach2/Blimp1 double-deficient LZ B cells, GSEA revealed a significant enrichment of c-Myc target genes, E2F target genes, and mTORC1 signaling genes, in that order (Fig. 3 C).

This was also demonstrated by flow cytometry analysis (expression levels of c-Myc, pRb, and pS6. Fig. 3 D).

Moreover, as expected, the mutant GC B cells were hyperproliferative, as assessed by 5-ethynyl-2′-deoxyuridine (EdU) pulse labeling (Fig. 3 E). These results, considering the previous demonstration that c-Myc–overexpressing and hyper-mTORC1 GC B cells manifest a bias toward the DZ (Ersching et al., 2017.

Finkin et al., 2019), like Bach2/Blimp1 double-deficient GC B cells, allowed us to hypothesize that the defective pro-memory in the mutant GC cells 5could result from anomalies of the mTORC1 and/or c-Myc pathways. Here, we focused our analysis on the mTORC1 pathway. To test this hypothesis, we first asked whether normalizing mTORC1 activity in Bach2/Blimp1 double-deficient GC cells could rescue development of pro-memory B cells and subsequent memory B cells.

We transferred Bach2f/fPrdm1f/fERT2cre B1-8hi B cells into rapamycin-resistant (MtorF2108L/F2108L) hosts (Ersching et al., 2017), deleted Bach2 and Prdm1 with tamoxifen, and then immunized the mice with NP-CGG/alum (Fig. 4 A). After immunization, the mice were treated with rapamycin to decrease mTORC1 activity in a transferred B cell–intrinsic manner.

As shown in Fig. 4 B, the dose of rapamycin used nearly normalized pS6 levels in the Bach2/Blimp1 double-deficient LZ B cells. The rapamycin treatment partially corrected the c-Myc overexpression and hyperproliferation observed in the Bach2/Blimp1 double-deficient B1-8hi B cells (Fig.

4 B), suggesting coexistence of mTORC1-dependent and -independent pathways to regulate c-Myc activities. In contrast to control vehicle treatment of Bach2/Blimp1 double-deficient B1-8hi B cells, upon rapamycin treatment, those mutant cells generated threefold higher numbers of IgG1+ memory B cells. The numbers of IgG1+CD73+ memory B cells were similarly increased (Fig.

4 C, right). Furthermore, the Fr.5:Fr.2 ratio was also increased upon rapamycin treatment (Fig. 4 D).

However, the memory B cells number upon rapamycin treatment did not reach those from wild-type B1-8hi B cells upon control vehicle injection (Fig. 4 C). Hence, we conclude that hyper-mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells is one of the mechanisms that cause defective development of memory B cells, although there must be other, currently unknown ones, as well.

In regard to GC B cells, the numbers were not significantly changed upon rapamycin treatment of Bach2/Blimp1 double-deficient B1-8hi B cells. Skewing of Bach2/Blimp1 double-deficient GC B cells toward the DZ was decreased upon rapamycin treatment, although a small enrichment was still observed (Fig. 4 C).

To further examine whether, in a wild-type setting, restraining mTORC1 activity could indeed facilitate differentiation of GC B cells to memory cells, we performed adoptive transfer experiments. For this, we conducted experiments in which two types of congenically marked B cells, rapamycin-sensitive (Mtor+/+) and rapamycin-resistant (MtorF2108L/F2108L) B1-8ge B cells, were cotransferred as a 1:1 mixture into rapamycin-resistant hosts (MtorF2108L/F2108L), which were immunized with NP-CGG/alum and then administered with rapamycin. As expected, rapamycin treatment led to a decrease in S6 phosphorylation in the transferred rapamycin-sensitive, but not rapamycin-resistant, B1-8ge GC B cells (Fig.

5 A). Upon rapamycin treatment, the number of rapamycin-sensitive NP+ GC B cells was decreased while the number of NP+ memory B cells was increased compared with their rapamycin-resistant counterparts, assessed by conventional flow cytometry analysis (Fig. 5 B).

To more directly demonstrate the transition from GC B cells to Fr.7 cells, we treated the immunized mice with EdU for 3 d (days 10–13) before analysis. In this setting, incorporation of EdU marks GC cells that divided during the treatment period and the resultant quiescent memory B cells (Fig. 5 C).

We previously confirmed that during this period, the majority of proliferating cells (>95%) are GC B cells and plasmablasts (Shinnakasu et al., 2016). Upon rapamycin treatment, the frequency of EdU+IgG1+ Fr.7 cells compared with GC cells was higher among the rapamycin-sensitive B1-8ge cells than the rapamycin-resistant ones, demonstrating rapamycin-mediated facilitation of the transition from GC to Fr.7 cells (Fig. 5 D).

Moreover, upon rapamycin treatment, the numbers of CD38−Bcl6hiCD69hi (Fr.2) and CD38intBcl6hi/intEfnb1+ (Fr.5) rapamycin-sensitive B1-8ge IgG1+ B cells were decreased and maintained, respectively. Thus, the ratio of Fr.5 to Fr.2 was increased (Fig. 5 E).

Together, we conclude that a relative enrichment in Fr.5 over Fr.2 cells is induced by rapamycin treatment, thereby facilitating the overall transition from GC B cells to memory B cells. The memory B cells generated in the presence of rapamycin were able to induce similar recall antibody responses to those generated in the absence of rapamycin, as assessed by adoptive transfer experiments (Fig. 5 F).

We next wished to examine why Fr.5, but not Fr.3 cells, can become pro-memory B cells. Since there were almost no differences in mTORC1 activity between Fr.5 and Fr.3 cells (Fig. S5 A), it appears that an mTORC1lo state is necessary but not sufficient for development of pro-memory B cells (Fr.5).

Thus, additional key properties must be required for development of these cells. Since one of crucial features of mature memory B cells is longevity, one straightforward possibility is that Fr.5 cells begin to acquire more survival activity. Supporting this idea, CD38intBcl6hi/intEfnb1+ (Fr.5) cells were less apoptotic compared with CD38−Bcl6hiCD69lo (Fr.3) cells as assessed by active caspase-3 staining (Fig.

6 A). Transcript data (Fig. 6 B) together with protein expression data (Fig.

6 C) demonstrated that Bcl2 expression was upregulated in Fr.5 cells compared with Fr.3 cells, and even more in pre-memory B cells (Fr.6). Similarly, we found that the cell surface BCR expression level was increased stepwise from Fr.3 to Fr.6 cells (Fig. 6 D).

We also observed a slight increase of IgG1 and Igα/β mRNA expression in Fr.5 over Fr.3 cells. Thus, regulation of both mRNA and protein levels seems to be operative. To examine whether Bcl2 family protein–mediated survival activity could impact the development of Fr.5 cells, we employed GC B cells with haploinsufficiency of Bim (Bcl2l11.

See Materials and methods), a counteracting factor against anti-apoptotic Bcl2-family members (O’Connor et al., 1998). Bcl2l11+/+ ERT2cre B1-8ge B cells and Bcl2l11f/+ERT2cre B1-8ge B cells were cotransferred as a 1:1 mixture into wild-type recipient mice, which were then immunized with NP-CGG/alum and treated with tamoxifen on day 8 (Fig. 6 E).

Bim mRNA expression was decreased to almost 50% of control levels after tamoxifen treatment in Bcl2l11f/+ GC B cells (Fig. 6 F). In this competitive setting, among the Fr.2/3/5/6 cells, the frequency was most significantly increased in Fr.5 and Fr.6 cells upon Bim haploinsufficiency (Fig.

6 G), although there was also a modest increase of Fr.3 cells. Consequently, the frequency of Bcl2l11f/+ NP+IgG1+CD73+ memory B cells was also increased (Fig. S5 B).

To examine the effects of surface BCR expression on survival, B1-8ge-flox/+ ERT2cre B cells were employed. For these particular experiments, we mixed these B cells and control B1-8ge/+ ERT2cre B cells at a 7:3 ratio and adoptively cotransferred them into recipient mice, which were then immunized with NP-CGG/alum (Fig. 6 H).

We injected tamoxifen on day 10 and examined surface BCR expression on day 12, demonstrating a significant decrease on Fr.5 cells derived from B1-8ge-flox/+ ERT2cre B cells (Fig. 6 I). To detect apoptotic cells in this experiment, we analyzed mixtures of Fr.5 and Fr.6 cells (CD38+Efnb1+) to acquire a sufficient number of cells for the assay.

As demonstrated in Fig. 6 J, concomitant with decreased surface BCR expression, there was a higher frequency of apoptotic (aCasp3+) cells among pro/pre-memory cells derived from B1-8ge-flox/+ ERT2cre B cells. Similarly, frequency of apoptotic cells among total LZ GC cells was enhanced upon BCR downregulation (Fig.

S5 C). A control experiment using Prdm1f/+B1-8ge/+ ERT2cre B cells showed that a nonspecific effect on apoptosis induced simply by Cre-mediated double-strand breaks was negligible (Fig. S5 D).

Together, stepwise increases of Bcl2 and surface BCR expression from pro-memory (Fr.5) cells to pre-memory (Fr.6) toward mature memory B cells are likely to contribute to their survival. It is still unclear what signals and processes in LZ GC cells initiate their differentiation toward long-lived memory B cells. Here, by focusing on key features for development of GC-derived memory precursors, we show that an mTORC1lo state is necessary to develop pro-memory B cells.

Since mTORC1lo LZ B cells receive weak T cell help and, as a result, have been thought to undergo apoptosis, this raises the question of how such pro-memory B cells are prevented from dying and able to differentiate into mature memory B cells. Our experiments suggest that the memory precursor B cells express higher levels of Bcl2 and surface BCR, thereby acquiring a survival advantage. We have already shown that Bach2hi LZ GC B cells are predisposed to differentiate into memory B cells (Shinnakasu et al., 2016), indicating that memory cell commitment already begins in a subset of GC B cells.

This memory-prone subset most likely corresponds to the Fr.5 (CD38intBcl6hi/intEfnb1+ pro-memory) cells. Indeed, expression of Bach2 in Fr.5 is higher than in Fr.2 cells (Fig. 2 A and Fig.

S3 B). Fr.6 (pre-memory B) cells appear to be undergoing a further developmental step toward mature memory B cells, manifested by further downregulation of Bcl6 (Fig. 1 B).

We found that mTORC1 has a marked effect on the ratio of memory-prone (Fr.5) to recycling-prone (Fr.2) GC B cell formation. Rapamycin treatment increased the proportion of Fr.5 cells and, conversely, hyperactivation of mTORC1 in the Bach2/Blimp1 double-deficient setting led to a relative increase in Fr.2 cells. Several nonmutually exclusive possibilities can be envisaged to explain why lower mTORC1 activity contributes to development of memory-prone cells.

Decay in mTORC1 activity as GC B cells proliferate in the DZ appears to be required for their timely return to the LZ (Ersching et al., 2017). Given the importance of LZ residency for memory differentiation (Bannard et al., 2013), one possibility is that LZ residency imposed by mTORC1lo could allow development of pro-memory B cells. Second, apart from this spatial requirement mediated through modulation of mTORC1, inhibition of mTORC1, as is seen during the generation of natural killer cell memory (O’Sullivan et al., 2015), may stimulate autophagy, thereby enhancing pro-memory B cell survival.

Finally, it is also well known that mTORC1 activity is suppressed in memory B cells (Boothby and Rickert, 2017). Such metabolic changes as the cells progress toward mature memory B cells thus appear to be initiated already in pro-memory cells, and this might be a necessary first step for generating mature memory B cells. The partial restoration of memory B cells by rapamycin treatment in Bach2/Blimp1 double-deficient GC cells suggests that, in addition to hyper-mTORC1 activity, other anomalies occur in mutant GC B cells in regard to memory differentiation.

One of them is likely the c-Myc overexpression, because of the following. First, indeed, in rapamycin-treated Bach2/Blimp1 double-deficient GC cells, overexpression of c-Myc and hyperproliferation were still observed to a significant extent (Fig. 4 B).

Second, c-Myc–overexpressing GC cells were reported to have a significant bias toward the DZ (Finkin et al., 2019). Considering the importance of LZ residency for memory differentiation (Bannard et al., 2013), overexpression of c-Myc is assumed to be detrimental to memory differentiation. Hence, we would propose that restraining both mTORC1-mediated metabolism and c-Myc–mediated cell-cycle progression is required to develop pro-memory B cells and that Bach2 is one of the critical regulators for suppressing both pathways.

Functionally, Bach2 is well known to act as a repressive guardian transcription factor (Igarashi et al., 2017). In regard to relationship between signaling and Bach2 expression, the mTORC1 activity and Bach2 expression appear to be mutually exclusive, because the BCR-induced AKT-mTORC1 inhibits Bach2 expression (Kometani et al., 2013), and Bach2 represses transcription of mTORC1 signaling molecules. Such a negative feedback loop is characteristic of “bistable” signal transduction circuits, which can operate in two stable formats.

This might take place between Fr.5 and Fr.2 cells. It should be mentioned that, from mTORC1 signaling molecule side, Bach2 is one of the transcription factors, and probably additional factors participate in transcriptional regulation on mTORC1 signaling genes. In addition to the connection between BCR signal and Bach2, considering the T cell data showing that ICOS and integrin αE are upregulated in Bach2lo T cells (Grant et al., 2020.

Sidwell et al., 2020), Bach2 might be involved in connecting the BCR signal to T cell help. For instance, Bach2lo LZ GC cells with high-affinity BCRs might modulate T/B interactions through adhesion status and coreceptor expression and affect the strength of T cell help. This might further downregulate Bach2, because we previously showed that strong T cell help depresses Bach2 expression (Shinnakasu et al., 2016).

After moving back into the LZ, apoptosis is generally thought to be the default pathway for LZ GC B cells. However, high-affinity cells are spared and positively selected after they encounter sufficient cognate T cell help (Allen et al., 2007. Victora and Nussenzweig, 2012).

These spared high-affinity cells correspond to Fr.2 cells, whereas the defaulting apoptotic LZ cells are likely to be Fr.3 cells. Indeed, among LZ GC cells, Fr.3 cells were most apoptotic. Here, we show that a small population of pro-memory B cells exists in the LZ and, despite apparently receiving weak T cell help, they are relatively resistant to apoptosis.

The inability of prior studies to detect such apoptosis-resistant LZ B cells is most likely due to the fact that the numbers of pro-memory cells are so limited (Mayer et al., 2017). Previous data using B cell–specific Bcl2-tg mice (Smith et al., 1994) or Bim knockout mice (Fischer et al., 2007) showed that such mice develop an enlarged memory B cell compartment. Recently, more detailed analysis using the same Bcl2-tg mice (Stewart et al., 2018) provided mechanistic insights into the above phenomenon.

First, in these mice, aberrant populations of seemingly quiescent cells arise that express markers of memory precursor cells. Second, overexpression of Bcl2 is not sufficient for DZ GC B cells with damaged BCRs to reach the LZ. Hence, in a physiological setting, it is reasonable to speculate that, after returning to the LZ in a Bcl2-independent manner, if Bcl2 expression is upregulated in some of the LZ GC B cells, they are better able to be rescued from apoptosis in the late G1 phase and to begin to differentiate into memory B cells.

Supporting this idea, we show here that among LZ GC cells, small numbers of pro-memory B cells (Fr.5), but not Fr.3 cells, begin to upregulate Bcl2, and that development of pro-memory B cells is facilitated by Bim haploinsufficiency. Because Bach2 expression in Fr.5 cells is similar to Fr.3 cells (Fig. S3 B), Bach2 appears not to be involved in such differential survival activity between Fr.5 and Fr.3 cells.

Rather, a Bach2-independent mechanism such as Bcl6 downregulation (discussed below) is likely to be operated, thereby allowing Fr.5 cells to survive enough to begin to differentiate into memory precursor cells. In contrast to Fr.5 cells (pro-memory), Fr.6 cells (pre-memory) apparently possess more survival activity (Fig. 6 A), possibly explaining the generation kinetics between Fr.5 and Fr.6 cells.

Fr.6 cells were more accumulated at later phases (days 14 and 20) during immune responses (Fig. S1 B). Induced downregulation of surface BCR expression in pro/pre-memory B cells resulted in increased apoptosis in the pro-memory B cells.

These results, together with the evidence that pro-memory B cells express higher surface BCR levels, lead us to propose that the BCR-mediated survival signal also plays a role in the development of pro-memory B cells. Based on the previous report that BCR ablation leads to cell death, which can be delayed by constitutive Bcl2 expression (Lam et al., 1997), we considered the possibility that downregulation of the BCR might decrease Bcl2 expression in pro/pre-memory B cells. However, we could not detect such a connection (data not shown).

In naive B cells, the constitutive PI3 kinase–Foxo1 pathway is known to replace the missing BCR-mediated survival signals (Srinivasan et al., 2009). Therefore, a question arises of how pro-memory B cells, despite being mTORC1lo (reflecting lower Akt activity), generate such a survival signal. Given that there is no enlarged GC phenotype in PTEN or Foxo1 knockout mice (Dominguez-Sola et al., 2015.

Inoue et al., 2017. Sander et al., 2015. Suzuki et al., 2003), one straightforward explanation might be that the quality and/or quantity of BCR-mediated survival signals differ between naive B cells and GC-derived memory B cells.

We provide evidence that downregulation of Bcl6 in pro-memory B cells could be one of the mechanisms for upregulation of Bcl2 and surface BCR. However, since the extent of Bcl6 downregulation in pro-memory B cells is small, such a slight change might not account for the observed upregulation of Bcl2 and surface BCR. Hence, our data cannot completely exclude the possibility that, particularly at the pro-memory B cell stage, other mechanisms might operate to initiate upregulation of Bcl2 and BCR.

In this case, it is likely that downregulation of Bcl6 acts as an amplification pathway for further upregulation of Bcl2 and surface BCR during differentiation toward mature memory B cells. In regard to this differentiation pathway, our data using Bcl6 haploinsufficiency are highly complementary to previous in vitro data that ectopic expression of Bcl6 in B cell cultures blocks the GC B cells from differentiating into memory B cells (Kuo et al., 2007). Together, it is likely that stepwise decreases in Bcl6 expression (pro-memory >.

Pre-memory >. Mature memory B cells) play a key role in memory B cell development. This raises the question of how is Bcl6 downregulated.

Three possibilities have already been reported. (1) upon strong BCR engagement, Erk-mediated degradation of Bcl6 (Niu et al., 1998). (2) transcriptional downregulation of Bcl6, mediated by CD40-activated IRF4 (Saito et al., 2007).

And (3) downregulation of Bcl6 by defective IL-21 signaling (Linterman et al., 2010). Among these, in regard to differentiation from GC to memory B cells, the final possibility seems to best fit with our observation that pro-memory B cells possess lower-affinity BCRs, thereby receiving less T cell help. In addition, as a transcriptional circuit–type regulation, the transcription factor Hhex, critical for memory B cell differentiation, has been recently reported to participate in downregulation of Bcl6 (Laidlaw et al., 2020).

In summary, this study provides important insights into the initial events for the fate decisions from GC to memory B cells. The modulation of cellular metabolism and survival play fundamental roles. Given the importance of GC-derived memory B cells for protection against heterologous virus reinfection (Leach et al., 2019.

Purtha et al., 2011), our findings may contribute to the development of efficient vaccination strategies. Single-cell suspensions of splenocytes were analyzed and sorted on a FACSCanto II (BD Biosciences) or a FACSAria II (BD Biosciences). Alexa647-active caspase-3, V500-B220, V450-Bcl6, BV786-CD138, BV510-CD38, V500-CD45.2, BV510-IgG1, PE-IgG1 antibodies, and BV786-streptavidin were purchased from BD Biosciences.

APC-eFluor780-B220, FITC-CD45.1, PE-CD45.1, APC-eFluor780-CD45.1, FITC-CD45.2, APC-eFluor780-CD45.2, APC-CD69, APC-eFluor780-CD69, eFluor450-CD73, PE-CD86, PerCP-Cy5.5-GL7 antibodies, PerCP-Cy5.5-streptavidin, PE-streptavidin, and APC-eFluor780-streptavidin were purchased from eBioscience. PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling.

C-Myc antibody was purchased from Abcam. PE-Bcl2 antibody was purchased from Miltenyi Biotec. Biotin-Efnb1 antibody was purchased from R&D Systems.

Alexa488-goat anti-rabbit IgG antibody was purchased from Thermo Fisher Scientific. For intracellular staining, the cells were fixed and permeabilized using a Foxp3 staining kit (eBioscience) for Bcl6, Bcl2, and pRb, a BD Cytofix/Cytoperm solution (BD Biosciences) for pS6 and active caspase-3, or a True-Nuclear Transcription Factor Staining Buffer Set (BioLegend) for c-Myc. APC-conjugated NP was prepared as described previously (Shinnakasu et al., 2016).

Incorporation of EdU was detected using a Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. We thank M.C. Nussenzweig (The Rockefeller University, New York, NY) for B1-8hi mice, T.

Okada (RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan) for Bcl6-YFP mice, G.D. Victora (The Rockefeller University) for MtorF2108L mice, and P.D. Burrows for critical reading of the manuscript.

This work was supported by grants from JSPS KAKENHI (JP17K08882 to T. Inoue. JP26221306 and JP19H01028 to T.

Kurosaki), the SENSHIN Medical Research Foundation (to T. Inoue), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. Inoue), and a research grant from Astellas Foundation for Research on Metabolic Disorders (to T.

Inoue and C. Kawai performed the experiments. T.

Inoue and T. Kurosaki designed the experiments. R.

Kitamura, and H. Fukuyama provided essential reagents. E.

Kawakami, N. Sax, and K. Yamashita performed bioinformatics analyses.

T. Inoue and T. Kurosaki wrote the manuscript..

Furosemide brands in canada

Furosemide
Lkc
Zovirax
Starlix
Hepcinat
Can cause heart attack
Upset stomach
Stuffy or runny nose
Back pain
Nausea
Headache
Over the counter
11h
4h
6h
18h
13h
Can you overdose
Online
Online
No
No
Yes
Where to buy
At cvs
RX pharmacy
Online Drugstore
On the market
At walgreens

That's a furosemide brands in canada lot of missed punch lines, important details in business meetings, sweet sentiments from a https://www.epide.fr/buy-furosemide-online-without-a-prescription/ loved one, cheerful bird songs and laughter from grandkids. In fact, your hearing aids will likely improve not just your ability to communicate but also your health. That's because hearing aids are linked to a reduced risk of cognitive decline and other health benefits. But hearing aids are a major purchase, so it's important to make sure you're prepared with these furosemide brands in canada 10 tips.

10 things we recommend before buying hearing aids A thorough hearing exam is a key step. 1. Hearing test The first thing you need is a thorough furosemide brands in canada hearing test and evaluation from a qualified hearing healthcare professional. Our consumer-reviewed directory can help you find a provider near you.

Hearing tests are easy and painless. Most insurance companies cover the cost of hearing tests, too furosemide brands in canada. 2. Priority list for your hearing needs Your hearing healthcare professional will do far more than just test your hearing on your first visit.

You will also have a discussion about your lifestyle furosemide brands in canada. Is listening to your favorite TV shows a big priority for you or would you rather prioritize being able to understand coworkers better?. Maybe you wish to stream music wirelessly through your hearing aids while taking walks or have easier one-on-one conversations at home. Whatever your priorities, communicate them clearly to your hearing care provider so they can more easily determine which products furosemide brands in canada are right for you.

3. Financial plan Unfortunately, hearing aids are not covered by Medicare or most third-party payers. While many people are working to change this, furosemide brands in canada hearing aids remain a major out-of-pocket expense. Help is available through financing programs, Vocational Rehabilitation if you are still working, grants and charitable organizations.

Do your homework so you can make a plan to pay for your hearing aids and stay within your budget. Your hearing care provider should give you furosemide brands in canada several options that will work for your hearing and your wallet. 4. Medical clearance If your hearing test indicates you may have a medical problem contributing to your hearing loss, make sure you see a physician to get a thorough work-up before pursuing hearing aids.

5 furosemide brands in canada. Realistic expectations Many hearing healthcare professionals think one of the most important factors in the success of their hearing aid patients is understanding that while today's hearing aids are amazing in their technological capabilities, they still cannot reproduce natural hearing. In excessively noisy environments, even normal hearing people have difficulty hearing every word clearly, and you may also experience some challenges even with the best hearing aids. Also, it furosemide brands in canada takes time to get used to hearing aids.

You may even find you hate your hearing aids at first, but eventually you'll find them invaluable. 6. An open furosemide brands in canada mind If you have preconceived notions about your hearing loss or what hearing aids are right for you, be ready to have those ideas challenged. Hearing aids have come a long way, technologically speaking, over the past decades, and you may be surprised to find the vast array of features and attractive styles that are available now.

Your hearing loss severity or type may mean only certain devices will work for you. Trust the process and the advice of your hearing furosemide brands in canada care professional. Don't just assume you'll want the tiniest or cheapest option. 7.

Motivation to hear better Your hearing healthcare professional will go to great lengths to make sure you succeed furosemide brands in canada with your new hearing aids, but you'll get better results if you put some effort into the process. Being engaged, providing valuable feedback about your experiences and keeping your follow-up appointments will help your provider make the right kinds of adjustments to your hearing aids so you get the most benefit. 8. Positive attitude As with most things in life, you will get the most from your hearing aids and your hearing healthcare provider if you stay positive.

Having a good attitude and furosemide brands in canada a sense of humor can help you get through most any challenge your hearing loss presents. 9. Support system Many new hearing aid wearers have been encouraged to take the leap by a family member or loved one who has become frustrated with longstanding hearing loss. Before you furosemide brands in canada start the process, discuss your decision with family, friends and even coworkers.

Advocating for yourself with them and asking for their support during your journey to better hearing will make you even more successful. 10. Last but not least, the furosemide brands in canada right hearing care professional Buying hearing aids isn't like buying a typical consumer good. These are highly sophisticated medical devices that require the expertise of a professional with experience in counseling and fitting.

A good working relationship is key, so be sure you feel comfortable with your provider and have a good rapport. Look for an office that keeps hours that are convenient for you so you can make your follow-up appointments easily.Hearing aids are complex devices, so it's important to understand when it's time to start considering buying a new pair.Hearing aids need replacing every furosemide brands in canada few years, depending on several factors. Aside from when your hearing aids are beyond repair as determined by a hearing specialist or audiologist, here are some other reasons to consider upgrading your hearing aids. You've had a change in hearing and/or health Just as our eyeglasses prescription changes with time, so too does our hearing.

You may find that your current furosemide brands in canada devices simply aren't powerful enough to help you. This may especially be the case if you now have severe-to-profound hearing loss but still use standard hearing aids. Instead, you might do better with stronger hearing aids, known as "power hearing aids." Likewise, a change in overall health can prompt the purchase of new devices. For example, arthritis might cause you furosemide brands in canada to have less dexterity in your fingers.

If you have in-the-ear hearing aids, the small battery door could be difficult to open with limited dexterity, so it might be a good idea to consider new behind-the-ear devices. Some models even come with rechargeable hearing aid batteries that require much less handling. Your hearing aids are more than 5 years old Most hearing aids last furosemide brands in canada between three and seven years. Many people wonder why they don't last longer, but the fact is that all hearing aids experience a lot of wear and tear.

Think about it. What other sophisticated electronic device do you furosemide brands in canada wear all day that's directly connected to you, working constantly?. Even if you take very good care of your device (such as frequent cleaning), continued natural exposure to moisture and ear wax has a damaging effect over time. Also, older devices simply don't function as efficiently as newer models and can even become obsolete.

Today's modern hearing aids are essentially tiny computers that run algorithms to constantly refine your hearing experience furosemide brands in canada. Depending on the hearing aid you buy, it likely uses advanced technology to. detect and minimize unnecessary background noise or wind noise detect and amplify the speaker directly in front of you be programmable via a smartphone app connect to external devices via Bluetooth You've made major lifestyle changes Sometimes, a lifestyle change is an excellent reason to get new hearing aids. You might realize that the technology level is no longer furosemide brands in canada meeting your needs or is outdated.

For example, you got a new phone and watch a lot of videos on it, but can't connect the sound directly to your hearing aids. Or, perhaps you're getting out and hiking a lot more than you used to, so you need hearing aids that can stand up to more rugged environments and are good at blocking wind noise. Or, on the other hand, if you don't get out as much as you used to, furosemide brands in canada a more basic model may work just fine for your needs. Your financial situation has improved Maybe when you bought your first pair of hearing aids a few years ago, you needed the most basic and economical option.

But if you can now afford more advanced devices, it might be time for an upgrade. Some people buy new hearing aids and keep their old ones as an extra set in case their new devices need repair. You've changed your attitude toward hearing aids Many people are very reluctant when they purchase their first hearing aids. In fact, it takes people up to 10 years on average to get hearing aids after first being diagnosed with hearing loss.

Additionally, it takes a while to learn what it means to hear your best, rather than just better. Thus, people who know about their needs and are more comfortable with hearing aids might want devices with different or more advanced settings since they have a better idea about what they want and need. If you're still not sure what to do, keep in mind that a qualified and compassionate hearing care provider can guide you. Find a consumer-reviewed hearing aid clinic near you with our directory of providers..

That's lowest price furosemide great! https://www.epide.fr/buy-furosemide-online-without-a-prescription/. According to the Hearing Review, people with hearing loss wait an average of seven years to get help. That's a lot of missed punch lines, important details in business meetings, sweet sentiments from a loved one, cheerful bird songs and laughter from grandkids. In fact, lowest price furosemide your hearing aids will likely improve not just your ability to communicate but also your health.

That's because hearing aids are linked to a reduced risk of cognitive decline and other health benefits. But hearing aids are a major purchase, so it's important to make sure you're prepared with these 10 tips. 10 things we recommend before buying hearing aids A thorough hearing lowest price furosemide exam is a key step. 1.

Hearing test The first thing you need is a thorough hearing test and evaluation from a qualified hearing healthcare professional. Our consumer-reviewed directory can lowest price furosemide help you find a provider near you. Hearing tests are easy and painless. Most insurance companies cover the cost of hearing tests, too.

2 lowest price furosemide. Priority list for your hearing needs Your hearing healthcare professional will do far more than just test your hearing on your first visit. You will also have a discussion about your lifestyle. Is listening to your favorite TV shows a big priority for you or would you rather prioritize being lowest price furosemide able to understand coworkers better?.

Maybe you wish to stream music wirelessly through your hearing aids while taking walks or have easier one-on-one conversations at home. Whatever your priorities, communicate them clearly to your hearing care provider so they can more easily determine which products are right for you. 3. Financial plan Unfortunately, hearing aids are not covered by Medicare or most third-party payers.

While many people are working to change this, hearing aids remain a major out-of-pocket expense. Help is available through financing programs, Vocational Rehabilitation if you are still working, grants and charitable organizations. Do your homework so you can make a plan to pay for your hearing aids and stay within your budget. Your hearing care provider should give you several options that will work for your hearing and your wallet.

4. Medical clearance If your hearing test indicates you may have a medical problem contributing to your hearing loss, make sure you see a physician to get a thorough work-up before pursuing hearing aids. 5. Realistic expectations Many hearing healthcare professionals think one of the most important factors in the success of their hearing aid patients is understanding that while today's hearing aids are amazing in their technological capabilities, they still cannot reproduce natural hearing.

In excessively noisy environments, even normal hearing people have difficulty hearing every word clearly, and you may also experience some challenges even with the best hearing aids. Also, it takes time to get used to hearing aids. You may even find you hate your hearing aids at first, but eventually you'll find them invaluable. 6.

An open mind If you have preconceived notions about your hearing loss or what hearing aids are right for you, be ready to have those ideas challenged. Hearing aids have come a long way, technologically speaking, over the past decades, and you may be surprised to find the vast array of features and attractive styles that are available now. Your hearing loss severity or type may mean only certain devices will work for you. Trust the process and the advice of your hearing care professional.

Don't just assume you'll want the tiniest or cheapest option. 7. Motivation to hear better Your hearing healthcare professional will go to great lengths to make sure you succeed with your new hearing aids, but you'll get better results if you put some effort into the process. Being engaged, providing valuable feedback about your experiences and keeping your follow-up appointments will help your provider make the right kinds of adjustments to your hearing aids so you get the most benefit.

8. Positive attitude As with most things in life, you will get the most from your hearing aids and your hearing healthcare provider if you stay positive. Having a good attitude and a sense of humor can help you get through most any challenge your hearing loss presents. 9.

Support system Many new hearing aid wearers have been encouraged to take the leap by a family member or loved one who has become frustrated with longstanding hearing loss. Before you start the process, discuss your decision with family, friends and even coworkers. Advocating for yourself with them and asking for their support during your journey to better hearing will make you even more successful. 10.

Last but not least, the right hearing care professional Buying hearing aids isn't like buying a typical consumer good. These are highly sophisticated medical devices that require the expertise of a professional with experience in counseling and fitting. A good working relationship is key, so be sure you feel comfortable with your provider and have a good rapport. Look for an office that keeps hours that are convenient for you so you can make your follow-up appointments easily.Hearing aids are complex devices, so it's important to understand when it's time to start considering buying a new pair.Hearing aids need replacing every few years, depending on several factors.

Aside from when your hearing aids are beyond repair as determined by a hearing specialist or audiologist, here are some other reasons to consider upgrading your hearing aids. You've had a change in hearing and/or health Just as our eyeglasses prescription changes with time, so too does our hearing. You may find that your current devices simply aren't powerful enough to help you. This may especially be the case if you now have severe-to-profound hearing loss but still use standard hearing aids.

Instead, you might do better with stronger hearing aids, known as "power hearing aids." Likewise, a change in overall health can prompt the purchase of new devices. For example, arthritis might cause you to have less dexterity in your fingers. If you have in-the-ear hearing aids, the small battery door could be difficult to open with limited dexterity, so it might be a good idea to consider new behind-the-ear devices. Some models even come with rechargeable hearing aid batteries that require much less handling.

Your hearing aids are more than 5 years old Most hearing aids last between three and seven years. Many people wonder why they don't last longer, but the fact is that all hearing aids experience a lot of wear and tear. Think about it. What other sophisticated electronic device do you wear all day that's directly connected to you, working constantly?.

Even if you take very good care of your device (such as frequent cleaning), continued natural exposure to moisture and ear wax has a damaging effect over time. Also, older devices simply don't function as efficiently as newer models and can even become obsolete. Today's modern hearing aids are essentially tiny computers that run algorithms to constantly refine your hearing experience. Depending on the hearing aid you buy, it likely uses advanced technology to.

detect and minimize unnecessary background noise or wind noise detect and amplify the speaker directly in front of you be programmable via a smartphone app connect to external devices via Bluetooth You've made major lifestyle changes Sometimes, a lifestyle change is an excellent reason to get new hearing aids. You might realize that the technology level is no longer meeting your needs or is outdated. For example, you got a new phone and watch a lot of videos on it, but can't connect the sound directly to your hearing aids. Or, perhaps you're getting out and hiking a lot more than you used to, so you need hearing aids that can stand up to more rugged environments and are good at blocking wind noise.

Or, on the other hand, if you don't get out as much as you used to, a more basic model may work just fine for your needs. Your financial situation has improved Maybe when you bought your first pair of hearing aids a few years ago, you needed the most basic and economical option. But if you can now afford more advanced devices, it might be time for an upgrade. Some people buy new hearing aids and keep their old ones as an extra set in case their new devices need repair.

You've changed your attitude toward hearing aids Many people are very reluctant when they purchase their first hearing aids. In fact, it takes people up to 10 years on average to get hearing aids after first being diagnosed with hearing loss. Additionally, it takes a while to learn what it means to hear your best, rather than just better. Thus, people who know about their needs and are more comfortable with hearing aids might want devices with different or more advanced settings since they have a better idea about what they want and need.

Does furosemide cause hyponatremia

REDWOOD CITY, Calif.--(BUSINESS WIRE)--Oct does furosemide cause hyponatremia. 16, 2020-- Guardant Health, Inc. (Nasdaq. GH) today announced it will report financial results for the third quarter 2020 after market close on Thursday, November 5, 2020.

Company management will be webcasting a corresponding conference call beginning at 1:30 p.m. Pacific Time / 4:30 p.m. Eastern Time. Live audio of the webcast will be available on the “Investors” section of the company website at.

Www.guardanthealth.com. The webcast will be archived and available for replay after the event. About Guardant Health Guardant Health is a leading precision oncology company focused on helping conquer cancer globally through use of its proprietary blood tests, vast data sets and advanced analytics. The Guardant Health Oncology Platform leverages capabilities to drive commercial adoption, improve patient clinical outcomes and lower healthcare costs across all stages of the cancer care continuum.

Guardant Health has launched liquid biopsy-based Guardant360®, Guardant360 CDx, and GuardantOMNI® tests for advanced stage cancer patients. These tests fuel development of its LUNAR program, which aims to address the needs of early stage cancer patients with neoadjuvant and adjuvant treatment selection, cancer survivors with surveillance, asymptomatic individuals eligible for cancer screening and individuals at a higher risk for developing cancer with early detection. View source version on businesswire.com. Https://www.businesswire.com/news/home/20201016005576/en/ Investor Contact.

Carrie Mendivilinvestors@guardanthealth.com Media Contact. Anna Czenepress@guardanthealth.com Courtney Carrollcourtney.carroll@uncappedcommunications.com Source. Guardant Health, Inc.REDWOOD CITY, Calif.--(BUSINESS WIRE)--Oct. 15, 2020-- Guardant Health, Inc.

(Nasdaq. GH) (“Guardant Health”), a leading precision oncology company focused on helping conquer cancer globally through use of its proprietary blood tests, vast data sets and advanced analytics, announced today the closings of an underwritten public offering of 7,700,000 shares of its common stock, which includes full exercise of the underwriter’s option to purchase 700,000 shares, at a public offering price of $102.00 per share, before deducting underwriting discounts and commissions, all of which were sold by SoftBank Investment Advisers. The initial closing of 7,000,000 shares occurred on October 9, 2020, and the closing of the underwriter’s option to purchase additional shares occurred today. Guardant Health did not sell any of its shares in the offering and did not receive any of the proceeds from the sale of shares in the offering by SoftBank Investment Advisers.

J.P. Morgan Securities LLC acted as sole book-running manager of the offering. The public offering was made pursuant to an automatic shelf registration statement on Form S-3 that was filed by Guardant Health with the U.S. Securities and Exchange Commission (the “SEC”) and automatically became effective upon filing.

A final prospectus supplement and accompanying prospectus relating to and describing the terms of the offering have been filed with the SEC and are available on the SEC’s website at www.sec.gov. Copies of the final prospectus supplement and accompanying prospectus may be obtained by contacting. J.P. Morgan Securities LLC, c/o Broadridge Financial Solutions, 1155 Long Island Avenue, Edgewood, NY 11717, or by telephone at (866) 803-9204, or by email at prospectus-eq_fi@jpmchase.com.

This press release shall not constitute an offer to sell or a solicitation of an offer to buy these securities, nor shall there be any sale of these securities in any state or other jurisdiction in which such offer, solicitation or sale would be unlawful prior to the registration or qualification under the securities laws of any such state or other jurisdiction. Source. Guardant Health, Inc. View source version on businesswire.com.

Https://www.businesswire.com/news/home/20201015005933/en/ Investors. Carrie Mendivilinvestors@guardanthealth.com Media. Anna Czenepress@guardanthealth.comSource. Guardant Health, Inc..

REDWOOD CITY, Calif.--(BUSINESS lowest price furosemide WIRE)--Oct. 16, 2020-- Guardant Health, Inc. (Nasdaq. GH) today announced it will report financial results for the third quarter 2020 after market close on Thursday, November 5, 2020. Company management will be webcasting a corresponding conference call beginning at 1:30 p.m.

Pacific Time / 4:30 p.m. Eastern Time. Live audio of the webcast will be available on the “Investors” section of the company website at. Www.guardanthealth.com. The webcast will be archived and available for replay after the event.

About Guardant Health Guardant Health is a leading precision oncology company focused on helping conquer cancer globally through use of its proprietary blood tests, vast data sets and advanced analytics. The Guardant Health Oncology Platform leverages capabilities to drive commercial adoption, improve patient clinical outcomes and lower healthcare costs across all stages of the cancer care continuum. Guardant Health has launched liquid biopsy-based Guardant360®, Guardant360 CDx, and GuardantOMNI® tests for advanced stage cancer patients. These tests fuel development of its LUNAR program, which aims to address the needs of early stage cancer patients with neoadjuvant and adjuvant treatment selection, cancer survivors with surveillance, asymptomatic individuals eligible for cancer screening and individuals at a higher risk for developing cancer with early detection. View source version on businesswire.com.

Https://www.businesswire.com/news/home/20201016005576/en/ Investor Contact. Carrie Mendivilinvestors@guardanthealth.com Media Contact. Anna Czenepress@guardanthealth.com Courtney Carrollcourtney.carroll@uncappedcommunications.com Source. Guardant Health, Inc.REDWOOD CITY, Calif.--(BUSINESS WIRE)--Oct. 15, 2020-- Guardant Health, Inc.

(Nasdaq. GH) (“Guardant Health”), a leading precision oncology company focused on helping conquer cancer globally through use of its proprietary blood tests, vast data sets and advanced analytics, announced today the closings of an underwritten public offering of 7,700,000 shares of its common stock, which includes full exercise of the underwriter’s option to purchase 700,000 shares, at a public offering price of $102.00 per share, before deducting underwriting discounts and commissions, all of which were sold by SoftBank Investment Advisers. The initial closing of 7,000,000 shares occurred on October 9, 2020, and the closing of the underwriter’s option to purchase additional shares occurred today. Guardant Health did not sell any of its shares in the offering and did not receive any of the proceeds from the sale of shares in the offering by SoftBank Investment Advisers. J.P.

Morgan Securities LLC acted as sole book-running manager of the offering. The public offering was made pursuant to an automatic shelf registration statement on Form S-3 that was filed by Guardant Health with the U.S. Securities and Exchange Commission (the “SEC”) and automatically became effective upon filing. A final prospectus supplement and accompanying prospectus relating to and describing the terms of the offering have been filed with the SEC and are available on the SEC’s website at www.sec.gov. Copies of the final prospectus supplement and accompanying prospectus may be obtained by contacting.

J.P. Morgan Securities LLC, c/o Broadridge Financial Solutions, 1155 Long Island Avenue, Edgewood, NY 11717, or by telephone at (866) 803-9204, or by email at prospectus-eq_fi@jpmchase.com. This press release shall not constitute an offer to sell or a solicitation of an offer to buy these securities, nor shall there be any sale of these securities in any state or other jurisdiction in which such offer, solicitation or sale would be unlawful prior to the registration or qualification under the securities laws of any such state or other jurisdiction. Source. Guardant Health, Inc.

View source version on businesswire.com. Https://www.businesswire.com/news/home/20201015005933/en/ Investors. Carrie Mendivilinvestors@guardanthealth.com Media. Anna Czenepress@guardanthealth.comSource. Guardant Health, Inc..

Can furosemide cause hyperkalemia

Artificial intelligence technologies are being increasingly relied upon in can furosemide cause hyperkalemia the healthcare domain, particularly when it comes furosemide canada pharmacy to decision support, precision medicine, and the improvement of the quality of care. Regarding primary care can furosemide cause hyperkalemia specifically, AI also represents an opportunity to assist with electronic health record documentation. A new study published in the Journal of American Medical Informatics Association this week shows that, although AI documentation assistants (or digital scribes) offer great potential in the primary care setting, they will need to be supervised by a human until strong evidence is available for their autonomous potential.

In workshops with primary care doctors, wrote researchers from the Australian Institute of Health Innovation, can furosemide cause hyperkalemia "There was consensus that consultations of the future would increasingly involve more automated and AI-supported systems. However, there were differing views on how this human-AI collaboration would work, what roles doctors and AI would take, and what tasks could be delegated to AI." HIMSS20 Digital Learn on-demand, earn credit, find products and solutions. Get Started can furosemide cause hyperkalemia >>.

WHY IT MATTERS Researchers worked with primary care doctors who use EHRs regularly for documentation purposes to understand their views on future AI documentation assistants. They identified three can furosemide cause hyperkalemia major themes that emerged from the discussions. Professional autonomy, human-AI collaboration and new models of care.

First, the doctors emphasized the importance of their ability to care for patients in their own can furosemide cause hyperkalemia way with the abilities AI technology provided."If they [patients] think that we're just getting suggestions from a computer, then maybe they can just get suggestions from a computer. I think it becomes more difficult to convince them that our recommendations are more valuable than what they can pick up on the internet," said one physician. They noted the need for a bottom-up approach to technology development, with a focus on delivering clear benefits to practice and workflow, and expressed fears around potential legal complications that could stem from working with an AI can furosemide cause hyperkalemia assistant.With regard to human-AI collaboration, doctors expressed a variety of viewpoints about what tasks could be delegated to AI.

Many believed that an AI system could assist with tasks such as documentation, referrals and other paperwork. Most said that AI can furosemide cause hyperkalemia systems would lack empathy. "GPs voiced several concerns, including some potential biases in patient data and system design, the time needed to fix the errors and train the system, challenges of dealing with complex cases, and the auditing of AI," wrote the researchers.

However, doctors also discussed how can furosemide cause hyperkalemia AI could help with emerging models of primary care, including preconsultation, mobile health and telehealth. THE LARGER TREND The question of reducing EHR-related clinician burnout has loomed large, with vendors and researchers trying to pinpoint major causes – and, in turn, potential solutions. AI has been raised as one such can furosemide cause hyperkalemia solution, with several major EHR vendors offering plans for incorporating the technology into their workflows.

But human input remains vital, as the new JAMIA study and other research has noted. AI could "bring back meaning and purpose in the practice of medicine while providing new levels of efficiency and accuracy," wrote Stanford researchers in a 2017 Journal of the can furosemide cause hyperkalemia American Medical Association study. But, they continued, physicians must "proactively guide, oversee, and monitor the adoption of artificial intelligence as a partner in patient care."ON THE RECORD"AI documentation assistants will likely ...

Be integral to can furosemide cause hyperkalemia the future primary care consultations. However, these technologies will still need to be supervised by a human until strong evidence for reliable autonomous performance is available. Therefore, different human-AI collaboration models will need to be can furosemide cause hyperkalemia designed and evaluated to ensure patient safety, quality of care, doctor safety, and doctor autonomy," wrote the Australian Institute for Health Innovation researchers.

Kat Jercich is senior editor of Healthcare IT News.Twitter. @kjercichHealthcare IT News is a HIMSS Media publication.Konica Minolta Healthcare Americas will pay $500,000 to settle a whistleblower case that alleged its Viztek electronic health record subsidiary had falsified data for can furosemide cause hyperkalemia certification tests.WHY IT MATTERSIn the qui tam complaint, filed in 2017 in U.S. District Court in New Jersey – where Konica Minolta is based – was filed by whistleblower Leighsa Wilson, who worked for two years at Viztek, best known for its PACS and imaging technologies, as a project manager for its EXA EHR product.In mid-2015, the complaint alleges, Viztek, which was in negotiations to be acquired by Konica Minolta, worked together with InfoGard Laboratories (which was then an ONC-authorized certification and testing body) to make false representations that the EHR software complied with requirements for certification – and qualified for receipt of incentive payments under the federal meaningful use program."To ensure that their product was certified and that their customers received incentive payments, Viztek and Konica Minolta.

(a) falsely attested to can furosemide cause hyperkalemia InfoGard that their software met the certification criteria. (b) hard-coded their software to pass certification testing requirements temporarily without ensuring that the software released to customers met certification criteria. And (c) caused their users to falsely attest to using a certified EHR technology, when their software could not support the applicable certification criteria in the field," according to the complaint, which also alleges that InfoGard can furosemide cause hyperkalemia "facilitated and participated in" these false attestations, "knowingly or with reckless disregard," certifying the EHR software despite its inability to meet ONC's certification criteria.The flaws in Viztek's software "not only rendered the system unreliable and unable to meet meaningful use standards, but the flaws also created a risk to patient health and safety.

Rather than spend the time and resources necessary to correct the flaws in its EHR software, the EHR defendants opted to do nothing."THE LARGER TRENDThis is only the most recent settlement of this type from health IT vendors accused of False Claims Act violations, of course.Most notable, was the case of eClinicalWorks, which was alleged by the Department of Justice to have falsely claimed meaningful use certification, to have neglected to have safety addressed issues in its software and to have paid kickbacks to clients. That case was settled in 2017 for $155 million.More recently, similar complaints were lodged against companies such as Practice can furosemide cause hyperkalemia Fusion and Greenway Health. They settled with DOJ for $145 million and $57 million, respectively."We will be unflagging in our efforts to preserve the accuracy and reliability of Americans’ health records and guard the public against corporate greed," said U.S.

Attorney for the District of Vermont Christina Nolan after can furosemide cause hyperkalemia the Greenway case this past year. "EHR companies should consider themselves on notice."ON THE RECORD"The lives of patients depend upon the information processed by electronic health records," said Wilson – who, as a qui tam whistleblower will receive 20% of the financial settlement – in a statement. "Functionality testing and subsequent certification must be performed and obtained through a reliable, measurable process.""Filing a qui tam lawsuit is a powerful and effective way to report problems with EHR software purchased with federal funds and get the problems fixed can furosemide cause hyperkalemia when they are ignored," said Luke Diamond, an associate at Phillips &.

Cohen. "The False Claims Act protects whistleblowers from job retaliation and offers rewards if the government recovers funds as a result of the qui tam case.""Our client can furosemide cause hyperkalemia was concerned about possible patient harm that can occur if EHR software isn't properly certified, so she stepped forward to inform the government about what she had witnessed," said Colette Matzzie, a partner and whistleblower attorney with Phillips &. Cohen, which brought the case.

"Ensuring that EHR software meets all governmental requirements is important to safeguard both patient care and federal funds."The Arc Madison Cortland in Oneida, New York, knows that there is can furosemide cause hyperkalemia a lack of providers that specialize in the intellectual/developmental disability field. Making the problem worse, not so many that understand dual diagnosis.THE PROBLEMWith COVID-19 can furosemide cause hyperkalemia minimizing the ability for individuals to receive face-to-face services with their providers, many patients are resorting to emergency department visits.Additionally, The Arc is in a rural area requiring travel to see a provider, and there is a lack of providers in the field. The population itself is underserved, with a lack of transportation to get to appointments.

Without the ability to institute telemedicine as a solution to these problems, the population supported can furosemide cause hyperkalemia by The Arc would have seen a lengthy (permanent?. ) pause for needed medical services.PROPOSALThe Arc this year received funding from the FCC to help provide telehealth services.“With this funding we can further treat patients, reduce crisis and allow for social distancing, which is imperative to our vulnerable population,” said Jackie Fahey, director of clinic services at The Arc Madison Cortland. €œWe could provide ongoing services to the individuals we serve can furosemide cause hyperkalemia to ensure there are no unnecessary emergency department visits.

This places less of a strain on our local emergency departments and unneeded additional costs.”With the purchase of tablets and headsets and telehealth services from vendor Doxy.me, The Arc was able to still provide medical care to its population of people with an I/DD. Additionally, eliminating emergency department visits also eliminates their exposure to COVID-19 and eases can furosemide cause hyperkalemia the burden of the ED providers who are overburdened right now.MARKETPLACEThere are many vendors of telemedicine technology and services on the health IT market today. Healthcare IT News recently compiled a comprehensive list of these vendors with detailed descriptions.

To read this special report, click here.MEETING THE CHALLENGE“When all of our locations were closed abruptly in the middle of March due to the COVID-19 pandemic, we needed to determine a way to quickly and easily implement a telehealth solution so that we were able to still support the individuals that we serve during the crisis, especially when many were under strict quarantine protocols for a variety of reasons,” Fahey explained.“We signed up immediately for the can furosemide cause hyperkalemia Doxy.me telehealth platform as it was a user-friendly platform that is HIPAA-compliant. The feature we liked about Doxy.me was that it is web-based, so nothing had to be downloaded and it could easily be used on a laptop, tablet or smartphone.”The Arc rolled out the technology initially with its mental health providers, who offer psychiatry/medication monitoring services, social work counseling and mental health counseling. More than half the organization’s enrollment is enrolled in one or all of these three services, so it was able to continue providing services to a can furosemide cause hyperkalemia large number of enrolled individuals.“We then began to roll the telehealth services out to nutrition, speech therapy, physical therapy and occupational therapy caseloads if individuals were appropriate to receive the service through telehealth,” Fahey said.RESULTSThe first success metric The Arc has been able to achieve with the technology is maintaining its utilization for mental health services.

When everything was running normal prior to COVID-19, The Arc’s mental health services made up about 25% of the services it provided on a monthly basis. With the implementation of telehealth services during the COVID-19 pandemic, the organization was able to achieve 20% of the services provided on a monthly basis.This has shown to staff that they have been able to still serve and respond to the needs of their psychiatry, social work and mental health counseling patients with can furosemide cause hyperkalemia minimal issues by implementing the telehealth technology.“The second success metric we have been able to achieve with the technology is we have been able to continue to receive referrals for our services and enroll new individuals into the services they need if the services are able to be completed via telehealth,” she said. €œBetween April, May and June, we have enrolled 16 new individuals into ongoing clinic services, which is right on par for our normal enrollment average per month.”USING FCC AWARD FUNDSThe Arc Madison Cortland was awarded $49,455 by the FCC earlier this year for laptop computers and headsets to provide remote consultations and treatment during the COVID-19 pandemic for psychological services, counseling, and occupational and physical therapy for people with developmental and other disabilities.“With the funds, we purchased headsets and tablets to allow the people we support to have access to medical appointments, along with physical therapy, occupational therapy and psychology appointments remotely,” Fahey explained.

€œThe technology enables us to continue to provide these services at a time when the can furosemide cause hyperkalemia people we support are unable to leave for traditional in-person appointments.“Because these are such uncertain times, and a time frame for when we may return to ‘normalcy’ is unknown, the technology allows us to continue delivering medical support without the concern of a pause in those services.”Twitter. @SiwickiHealthITEmail the writer. Bill.siwicki@himss.orgHealthcare IT News is a HIMSS Media publication.HIMSSCast host Jonah Comstock convenes a panel of HIMSS Media editors – HITN Senior can furosemide cause hyperkalemia Editor Kat Jercich, MobiHealthNews Associate Editor Dave Muoio and HFN Associate Editor Jeff Lagasse – to discuss recent delivery slowdowns at the Post Office and how they have and haven't affected healthcare stakeholders, including startups and patients.

The team also looks into the broader trend of the politicization of traditionally apolitical government agencies and how that could affect public faith in COVID-19 treatments or vaccines.More about this episode:USPS service delays are hitting some mail-order pharmacies and telehealth platforms harder than othersMail delays may affect medication supply for nearly 1 in 4 Americans over 50Postmaster General Louis DeJoy's full testimony (C-SPAN)The Package Coalition homepageThe Trump administration this week asked the U.S. Supreme Court to reverse a lower court ruling that allowed can furosemide cause hyperkalemia for mail-order and telemedicine abortion during the COVID-19 crisis. U.S.

Food and Drug Administration regulations require mifepristone, which is used in medication abortion, to be dispensed at a clinic, hospital or can furosemide cause hyperkalemia medical office. In June, U.S. District Judge for the District of Maryland Theodore Chuang blocked the requirements during the pandemic, finding them to be a "substantial obstacle." Mifepristone, in combination with misoprostol, is can furosemide cause hyperkalemia FDA-approved for abortions up to ten weeks' gestation.

In 2017, a New England Journal of Medicine article argued against the FDA regulations for mifepristone given the drug's safety record. WHY IT MATTERS Acting Solicitor General Jeffrey B can furosemide cause hyperkalemia. Wall applied for a stay of Chuang's injunction on Wednesday as the case makes its way through the lower courts, arguing that the regulations do not represent an undue burden.

"The can furosemide cause hyperkalemia safety requirements here concern only medication abortions using Mifeprex, which is approved for use only during the first ten weeks of pregnancy. They have no effect on the availability of surgical abortions, a method that this Court has treated as safe for women," wrote Wall. Reproductive rights groups spoke out against the move, noting that people can furosemide cause hyperkalemia of color are disproportionately affected both by abortion restrictions and by the COVID-19 pandemic.

"Black, Brown, Indigenous people and people of color are already dying/getting sick at disproportionate rates from COVID-19," said All Above All* on Twitter. "The Trump-Pence admin is trying to make this worse by asking SCOTUS to require people face unnecessary risk just to get abortion care." "The FDA’s in-person requirements on mifepristone subject patients to can furosemide cause hyperkalemia unnecessary exposure to a deadly virus, and two federal courts have already rejected the Trump administration’s argument. Forcing patients to travel to a health center to access the safe, effective medication they need especially hurts people of color and people with low-incomes, who already face more barriers to care," said Planned Parenthood Federation of America President and CEO Alexis McGill-Johnson in a statement.THE LARGER TREND The COVID-19 pandemic has exacerbated many existing barriers to care, including for reproductive health services.

"We’ve seen the undue burden and hardship these restrictions create during COVID-19, especially in communities hit hardest can furosemide cause hyperkalemia by the pandemic," said Skye Perryman, chief legal officer at the American College of Obstetricians and Gynecologists, a co-plaintiff in the telemedicine case, to Healthcare IT News. In response to the July ruling, some abortion providers reportedly moved to delivering mifepristone by mail. Still, others faced state laws that restricted can furosemide cause hyperkalemia the provision of abortion via telemedicine.And as Dr.

Jacquelyn Yeh from Physicians from Reproductive Health pointed out in July, telemedicine itself involves hurdles such as broadband access and privacy concerns. It remains to be seen whether the Supreme Court can furosemide cause hyperkalemia will grant the Trump administration's request. ON THE RECORD "As COVID-19 ravages Black, Latino, Indigenous, and other communities of color across the country, the Trump administration should be aiming to keep us healthy – not moving forward with an agenda to endanger people who seek abortion," said McGill-Johnson.

Kat Jercich is senior editor of Healthcare IT News.Twitter. @kjercichHealthcare IT News is a HIMSS Media publication..

Artificial intelligence technologies are being increasingly relied upon in the healthcare domain, particularly when it comes to decision support, precision medicine, lowest price furosemide and the improvement of the quality of care. Regarding primary care specifically, AI lowest price furosemide also represents an opportunity to assist with electronic health record documentation. A new study published in the Journal of American Medical Informatics Association this week shows that, although AI documentation assistants (or digital scribes) offer great potential in the primary care setting, they will need to be supervised by a human until strong evidence is available for their autonomous potential. In workshops with primary care doctors, wrote researchers from the Australian Institute of Health Innovation, "There was consensus that consultations of the future lowest price furosemide would increasingly involve more automated and AI-supported systems. However, there were differing views on how this human-AI collaboration would work, what roles doctors and AI would take, and what tasks could be delegated to AI." HIMSS20 Digital Learn on-demand, earn credit, find products and solutions.

Get Started >> lowest price furosemide. WHY IT MATTERS Researchers worked with primary care doctors who use EHRs regularly for documentation purposes to understand their views on future AI documentation assistants. They identified three major themes that emerged from lowest price furosemide the discussions. Professional autonomy, human-AI collaboration and new models of care. First, the doctors emphasized the importance of their ability to care for patients in their own way with the abilities AI technology provided."If they [patients] think that we're just getting suggestions from a computer, then maybe lowest price furosemide they can just get suggestions from a computer.

I think it becomes more difficult to convince them that our recommendations are more valuable than what they can pick up on the internet," said one physician. They noted the need for a bottom-up approach to technology development, with a focus on delivering clear benefits to practice and workflow, and expressed fears lowest price furosemide around potential legal complications that could stem from working with an AI assistant.With regard to human-AI collaboration, doctors expressed a variety of viewpoints about what tasks could be delegated to AI. Many believed that an AI system could assist with tasks such as documentation, referrals and other paperwork. Most said lowest price furosemide that AI systems would lack empathy. "GPs voiced several concerns, including some potential biases in patient data and system design, the time needed to fix the errors and train the system, challenges of dealing with complex cases, and the auditing of AI," wrote the researchers.

However, doctors also discussed how AI could help with emerging models of primary care, including preconsultation, mobile health and lowest price furosemide telehealth. THE LARGER TREND The question of reducing EHR-related clinician burnout has loomed large, with vendors and researchers trying to pinpoint major causes – and, in turn, potential solutions. AI has been raised as one such solution, with several major EHR vendors offering plans for incorporating lowest price furosemide the technology into their workflows. But human input remains vital, as the new JAMIA study and other research has noted. AI could "bring back meaning and purpose in the practice of lowest price furosemide medicine while providing new levels of efficiency and accuracy," wrote Stanford researchers in a 2017 Journal of the American Medical Association study.

But, they continued, physicians must "proactively guide, oversee, and monitor the adoption of artificial intelligence as a partner in patient care."ON THE RECORD"AI documentation assistants will likely ... Be integral to the future primary lowest price furosemide care consultations. However, these technologies will still need to be supervised by a human until strong evidence for reliable autonomous performance is available. Therefore, different human-AI collaboration models will need to be designed and evaluated to ensure patient safety, quality of care, doctor lowest price furosemide safety, and doctor autonomy," wrote the Australian Institute for Health Innovation researchers. Kat Jercich is senior editor of Healthcare IT News.Twitter.

@kjercichHealthcare IT News is a HIMSS Media publication.Konica Minolta Healthcare Americas will pay $500,000 to settle a whistleblower case that alleged its Viztek electronic health record subsidiary had falsified data for certification tests.WHY IT MATTERSIn the qui tam complaint, lowest price furosemide filed in 2017 in U.S. District Court in New Jersey – where Konica Minolta is based – was filed by whistleblower Leighsa Wilson, who worked for two years at Viztek, best known for its PACS and imaging technologies, as a project manager for its EXA EHR product.In mid-2015, the complaint alleges, Viztek, which was in negotiations to be acquired by Konica Minolta, worked together with InfoGard Laboratories (which was then an ONC-authorized certification and testing body) to make false representations that the EHR software complied with requirements for certification – and qualified for receipt of incentive payments under the federal meaningful use program."To ensure that their product was certified and that their customers received incentive payments, Viztek and Konica Minolta. (a) falsely attested to lowest price furosemide InfoGard that their software met the certification criteria. (b) hard-coded their software to pass certification testing requirements temporarily without ensuring that the software released to customers met certification criteria. And (c) caused their users to falsely attest to using a certified EHR technology, when their software could not support the applicable certification criteria in the field," according to the complaint, which also alleges that InfoGard "facilitated and participated in" these false attestations, "knowingly or with reckless disregard," certifying the EHR software despite its inability to meet ONC's certification criteria.The flaws in Viztek's software "not only rendered lowest price furosemide the system unreliable and unable to meet meaningful use standards, but the flaws also created a risk to patient health and safety.

Rather than spend the time and resources necessary to correct the flaws in its EHR software, the EHR defendants opted to do nothing."THE LARGER TRENDThis is only the most recent settlement of this type from health IT vendors accused of False Claims Act violations, of course.Most notable, was the case of eClinicalWorks, which was alleged by the Department of Justice to have falsely claimed meaningful use certification, to have neglected to have safety addressed issues in its software and to have paid kickbacks to clients. That case was settled in 2017 for $155 million.More recently, similar complaints were lodged against companies such as Practice Fusion and Greenway lowest price furosemide Health. They settled with DOJ for $145 million and $57 million, respectively."We will be unflagging in our efforts to preserve the accuracy and reliability of Americans’ health records and guard the public against corporate greed," said U.S. Attorney for the lowest price furosemide District of Vermont Christina Nolan after the Greenway case this past year. "EHR companies should consider themselves on notice."ON THE RECORD"The lives of patients depend upon the information processed by electronic health records," said Wilson – who, as a qui tam whistleblower will receive 20% of the financial settlement – in a statement.

"Functionality testing and subsequent certification must be performed and obtained through a reliable, lowest price furosemide measurable process.""Filing a qui tam lawsuit is a powerful and effective way to report problems with EHR software purchased with federal funds and get the problems fixed when they are ignored," said Luke Diamond, an associate at Phillips &. Cohen. "The False Claims Act protects whistleblowers from job retaliation and offers rewards if the government recovers funds as a result of the qui tam case.""Our client was concerned about possible patient lowest price furosemide harm that can occur if EHR software isn't properly certified, so she stepped forward to inform the government about what she had witnessed," said Colette Matzzie, a partner and whistleblower attorney with Phillips &. Cohen, which brought the case. "Ensuring that EHR software meets all governmental requirements is important to safeguard both patient care and federal funds."The Arc Madison Cortland in Oneida, New York, knows that there is a lack of providers that specialize in the intellectual/developmental lowest price furosemide disability field.

Making the problem worse, not so many that understand dual diagnosis.THE PROBLEMWith COVID-19 minimizing the ability for individuals to receive face-to-face services with their providers, many patients are lowest price furosemide resorting to emergency department visits.Additionally, The Arc is in a rural area requiring travel to see a provider, and there is a lack of providers in the field. The population itself is underserved, with a lack of transportation to get to appointments. Without the ability to institute telemedicine as a solution to these problems, lowest price furosemide the population supported by The Arc would have seen a lengthy (permanent?. ) pause for needed medical services.PROPOSALThe Arc this year received funding from the FCC to help provide telehealth services.“With this funding we can further treat patients, reduce crisis and allow for social distancing, which is imperative to our vulnerable population,” said Jackie Fahey, director of clinic services at The Arc Madison Cortland. €œWe could provide ongoing services to the individuals we serve to ensure lowest price furosemide there are no unnecessary emergency department visits.

This places less of a strain on our local emergency departments and unneeded additional costs.”With the purchase of tablets and headsets and telehealth services from vendor Doxy.me, The Arc was able to still provide medical care to its population of people with an I/DD. Additionally, eliminating emergency department visits also eliminates their exposure to COVID-19 and eases the burden of the ED providers who are overburdened right now.MARKETPLACEThere are many vendors of telemedicine technology and lowest price furosemide services on the health IT market today. Healthcare IT News recently compiled a comprehensive list of these vendors with detailed descriptions. To read this special report, click here.MEETING THE CHALLENGE“When all of our locations were closed abruptly in the middle of March due to the COVID-19 pandemic, we needed to determine a way to quickly and easily implement a telehealth solution so that we were able to still support the individuals that we serve during the crisis, especially when many were under strict quarantine protocols for a variety of reasons,” Fahey explained.“We signed up immediately for the Doxy.me telehealth platform as it lowest price furosemide was a user-friendly platform that is HIPAA-compliant. The feature we liked about Doxy.me was that it is web-based, so nothing had to be downloaded and it could easily be used on a laptop, tablet or smartphone.”The Arc rolled out the technology initially with its mental health providers, who offer psychiatry/medication monitoring services, social work counseling and mental health counseling.

More than half the organization’s enrollment is enrolled in one or all of these three services, so it was able to continue providing services to a large number of enrolled individuals.“We then began to roll the telehealth services out to nutrition, speech therapy, physical therapy and occupational therapy caseloads if individuals were appropriate to receive the service through telehealth,” Fahey said.RESULTSThe first success metric The Arc has been able to achieve with the technology is maintaining its utilization for mental health lowest price furosemide services. When everything was running normal prior to COVID-19, The Arc’s mental health services made up about 25% of the services it provided on a monthly basis. With the implementation of telehealth services during the COVID-19 pandemic, the organization was able to achieve 20% of the services provided on a monthly basis.This has shown to staff that they have been able to still serve and respond to the needs of their psychiatry, social work and mental health counseling patients with minimal issues lowest price furosemide by implementing the telehealth technology.“The second success metric we have been able to achieve with the technology is we have been able to continue to receive referrals for our services and enroll new individuals into the services they need if the services are able to be completed via telehealth,” she said. €œBetween April, May and June, we have enrolled 16 new individuals into ongoing clinic services, which is right on par for our normal enrollment average per month.”USING FCC AWARD FUNDSThe Arc Madison Cortland was awarded $49,455 by the FCC earlier this year for laptop computers and headsets to provide remote consultations and treatment during the COVID-19 pandemic for psychological services, counseling, and occupational and physical therapy for people with developmental and other disabilities.“With the funds, we purchased headsets and tablets to allow the people we support to have access to medical appointments, along with physical therapy, occupational therapy and psychology appointments remotely,” Fahey explained. €œThe technology enables us to continue to provide these services at a time when the people we support are unable to leave for traditional in-person appointments.“Because these are such uncertain times, and a time frame for when we may return to ‘normalcy’ is unknown, the technology allows us to continue delivering medical support lowest price furosemide without the concern of a pause in those services.”Twitter.

@SiwickiHealthITEmail the writer. Bill.siwicki@himss.orgHealthcare IT News is a HIMSS Media publication.HIMSSCast host Jonah Comstock convenes a panel of HIMSS Media editors – HITN Senior Editor Kat Jercich, MobiHealthNews Associate Editor Dave Muoio and HFN Associate Editor Jeff Lagasse – to discuss recent delivery slowdowns at the Post Office and how they have and haven't affected healthcare stakeholders, lowest price furosemide including startups and patients. The team also looks into the broader trend of the politicization of traditionally apolitical government agencies and how that could affect public faith in COVID-19 treatments or vaccines.More about this episode:USPS service delays are hitting some mail-order pharmacies and telehealth platforms harder than othersMail delays may affect medication supply for nearly 1 in 4 Americans over 50Postmaster General Louis DeJoy's full testimony (C-SPAN)The Package Coalition homepageThe Trump administration this week asked the U.S. Supreme Court to reverse a lower court ruling that allowed lowest price furosemide for mail-order and telemedicine abortion during the COVID-19 crisis. U.S.

Food and Drug Administration regulations require mifepristone, which is used in medication abortion, to lowest price furosemide be dispensed at a clinic, hospital or medical office. In June, U.S. District Judge for the District of Maryland Theodore Chuang blocked the requirements during the pandemic, finding them to be a "substantial obstacle." Mifepristone, in combination with lowest price furosemide misoprostol, is FDA-approved for abortions up to ten weeks' gestation. In 2017, a New England Journal of Medicine article argued against the FDA regulations for mifepristone given the drug's safety record. WHY IT MATTERS lowest price furosemide Acting Solicitor General Jeffrey B.

Wall applied for a stay of Chuang's injunction on Wednesday as the case makes its way through the lower courts, arguing that the regulations do not represent an undue burden. "The safety requirements here concern only medication abortions using Mifeprex, which is approved for use only during the lowest price furosemide first ten weeks of pregnancy. They have no effect on the availability of surgical abortions, a method that this Court has treated as safe for women," wrote Wall. Reproductive rights groups spoke out lowest price furosemide against the move, noting that people of color are disproportionately affected both by abortion restrictions and by the COVID-19 pandemic. "Black, Brown, Indigenous people and people of color are already dying/getting sick at disproportionate rates from COVID-19," said All Above All* on Twitter.

"The Trump-Pence admin is trying to make this worse by asking SCOTUS to require people face unnecessary risk just to get abortion care." "The FDA’s in-person requirements on mifepristone subject patients to unnecessary exposure to a deadly virus, and two federal lowest price furosemide courts have already rejected the Trump administration’s argument. Forcing patients to travel to a health center to access the safe, effective medication they need especially hurts people of color and people with low-incomes, who already face more barriers to care," said Planned Parenthood Federation of America President and CEO Alexis McGill-Johnson in a statement.THE LARGER TREND The COVID-19 pandemic has exacerbated many existing barriers to care, including for reproductive health services. "We’ve seen the undue burden and hardship these restrictions create during COVID-19, especially in communities hit hardest by the pandemic," said Skye Perryman, chief legal officer at the American College of Obstetricians and Gynecologists, a co-plaintiff in the telemedicine lowest price furosemide case, to Healthcare IT News. In response to the July ruling, some abortion providers reportedly moved to delivering mifepristone by mail. Still, lowest price furosemide others faced state laws that restricted the provision of abortion via telemedicine.And as Dr.

Jacquelyn Yeh from Physicians from Reproductive Health pointed out in July, telemedicine itself involves hurdles such as broadband access and privacy concerns. It remains to be seen whether the Supreme Court lowest price furosemide will grant the Trump administration's request. ON THE RECORD "As COVID-19 ravages Black, Latino, Indigenous, and other communities of color across the country, the Trump administration should be aiming to keep us healthy – not moving forward with an agenda to endanger people who seek abortion," said McGill-Johnson. Kat Jercich is senior editor of Healthcare IT News.Twitter. @kjercichHealthcare IT News is a HIMSS Media publication..

Furosemide for horses

September 10, 2020U.S furosemide for horses. Department of Labor Cites Christus Shreveport-Bossier Health System For Failing to Protect Employees from the Coronavirus SHREVEPORT, LA – The U.S. Department of Labor's Occupational Safety and Health Administration (OSHA) has cited furosemide for horses Christus Shreveport-Bossier Health System in Shreveport, Louisiana, for failing to ensure employees wore proper protective equipment. OSHA has proposed $13,494 in penalties, the maximum allowed by law for a serious citation. OSHA opened a coronavirus–related investigation after receiving reports of furosemide for horses employee exposure.

The agency found that emergency facility employees often shared used protective gowns or did not have protective gowns to wear while treating patients. "Employers, especially those within the healthcare industry, must comply with existing standards to help ensure workers' safety amidst the coronavirus pandemic," said OSHA Baton Rouge Area Director Roderic M. Chube. "Healthcare workers must be provided proper personal protective equipment to limit the spread of the virus." The company has 15 business days from receipt of the citation and penalties to comply, request an informal conference with OSHA's area director, or contest the findings before the independent Occupational Safety and Health Review Commission. Employers with questions on compliance with OSHA standards should contact their local OSHA office for guidance and assistance at 800-321-OSHA (6742).

OSHA's COVID-19 response webpage offers extensive resources for addressing safety and health hazards during the evolving coronavirus pandemic. Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees. OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education and assistance. For more information, visit http://www.osha.gov. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States.

Improve working conditions. Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact. Megan Sweeney, 202-693-4661, Sweeney.Megan.P@dol.gov Release Number.

20-1699-DAL U.S. Department of Labor news materials are accessible at http://www.dol.gov. The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).September 10, 2020U.S. Department of Labor Cites Smithfield Packaged Meats Corp.For Failing to Protect Employees from Coronavirus SIOUX FALLS, SD – The U.S.

Department of Labor's Occupational Safety and Health Administration (OSHA) has cited Smithfield Packaged Meats Corp. In Sioux Falls, South Dakota, for failing to protect employees from exposure to the coronavirus. OSHA proposed a penalty of $13,494, the maximum allowed by law. Based on a coronavirus-related inspection, OSHA cited the company for one violation of the general duty clause for failing to provide a workplace free from recognized hazards that can cause death or serious harm. At least 1,294 Smithfield workers contracted coronavirus, and four employees died from the virus in the spring of 2020.

€œEmployers must quickly implement appropriate measures to protect their workers' safety and health,” said OSHA Sioux Falls Area Director Sheila Stanley. €œEmployers must meet their obligations and take the necessary actions to prevent the spread of coronavirus at their worksite.” OSHA guidance details proactive measures employers can take to protect workers from the coronavirus, such as social distancing measures and the use of physical barriers, face shields and face coverings when employees are unable to physically distance at least 6 feet from each other. OSHA guidance also advises that employers should provide safety and health information through training, visual aids, and other means to communicate important safety warnings in a language their workers understand. Smithfield has 15 business days from receipt of the citation and penalty to comply, request an informal conference with OSHA's area director or contest the findings before the independent Occupational Safety and Health Review Commission. Employers with questions on compliance with OSHA standards should contact their local OSHA office for guidance and assistance at 800-321-OSHA (6742).

OSHA's coronavirus response webpage offers extensive resources for addressing safety and health hazards during the evolving coronavirus pandemic. Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees. OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education and assistance. For more information, visit https://www.osha.gov. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States.

Improve working conditions. Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact. Megan Sweeney, 202-693-4661, sweeney.megan.p@dol.gov Release Number.

20-1684-NAT U.S. Department of Labor news materials are accessible at http://www.dol.gov. The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

September 10, lowest price furosemide 2020U.S. Department of Labor Cites Christus Shreveport-Bossier Health System For Failing to Protect Employees from the Coronavirus SHREVEPORT, LA – The U.S. Department of Labor's Occupational Safety and Health Administration lowest price furosemide (OSHA) has cited Christus Shreveport-Bossier Health System in Shreveport, Louisiana, for failing to ensure employees wore proper protective equipment. OSHA has proposed $13,494 in penalties, the maximum allowed by law for a serious citation. OSHA opened lowest price furosemide a coronavirus–related investigation after receiving reports of employee exposure.

The agency found that emergency facility employees often shared used protective gowns or did not have protective gowns to wear while treating patients. "Employers, especially those within the healthcare industry, must comply with existing standards to help ensure workers' safety amidst the coronavirus pandemic," said OSHA Baton Rouge Area Director Roderic M. Chube. "Healthcare workers must be provided proper personal protective equipment to limit the spread of the virus." The company has 15 business days from receipt of the citation and penalties to comply, request an informal conference with OSHA's area director, or contest the findings before the independent Occupational Safety and Health Review Commission. Employers with questions on compliance with OSHA standards should contact their local OSHA office for guidance and assistance at 800-321-OSHA (6742).

OSHA's COVID-19 response webpage offers extensive resources for addressing safety and health hazards during the evolving coronavirus pandemic. Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees. OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education and assistance. For more information, visit http://www.osha.gov. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States.

Improve working conditions. Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact. Megan Sweeney, 202-693-4661, Sweeney.Megan.P@dol.gov Release Number.

20-1699-DAL U.S. Department of Labor news materials are accessible at http://www.dol.gov. The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).September 10, 2020U.S. Department of Labor Cites Smithfield Packaged Meats Corp.For Failing to Protect Employees from Coronavirus SIOUX FALLS, SD – The U.S.

Department of Labor's Occupational Safety and Health Administration (OSHA) has cited Smithfield Packaged Meats Corp. In Sioux Falls, South Dakota, for failing to protect employees from exposure to the coronavirus. OSHA proposed a penalty of $13,494, the maximum allowed by law. Based on a coronavirus-related inspection, OSHA cited the company for one violation of the general duty clause for failing to provide a workplace free from recognized hazards that can cause death or serious harm. At least 1,294 Smithfield workers contracted coronavirus, and four employees died from the virus in the spring of 2020.

€œEmployers must quickly implement appropriate measures to protect their workers' safety and health,” said OSHA Sioux Falls Area Director Sheila Stanley. €œEmployers must meet their obligations and take the necessary actions to prevent the spread of coronavirus at their worksite.” OSHA guidance details proactive measures employers can take to protect workers from the coronavirus, such as social distancing measures and the use of physical barriers, face shields and face coverings when employees are unable to physically distance at least 6 feet from each other. OSHA guidance also advises that employers should provide safety and health information through training, visual aids, and other means to communicate important safety warnings in a language their workers understand. Smithfield has 15 business days from receipt of the citation and penalty to comply, request an informal conference with OSHA's area director or contest the findings before the independent Occupational Safety and Health Review Commission. Employers with questions on compliance with OSHA standards should contact their local OSHA office for guidance and assistance at 800-321-OSHA (6742).

OSHA's coronavirus response webpage offers extensive resources for addressing safety and health hazards during the evolving coronavirus pandemic. Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees. OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education and assistance. For more information, visit https://www.osha.gov. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States.

Improve working conditions. Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact. Megan Sweeney, 202-693-4661, sweeney.megan.p@dol.gov Release Number.

20-1684-NAT U.S. Department of Labor news materials are accessible at http://www.dol.gov. The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

Furosemide mg

€œI was just scared that I’m not going to be able furosemide mg to see it through, that I’m going to get sick — you just feel so exhausted and it’s just a lot.”The Mountain West, which for months avoided the worst of the pandemic, has rapidly devolved into one of the most alarming hot spots in a country that recorded its eight millionth confirmed case on Thursday, a day when more than 65,000 cases were announced nationwide, the most in a single day since July.Seventeen states, including many in the Mountain West, have added more cases in the past week than any other week of the pandemic. And the spread through sparsely populated areas of rural America has created problems in small towns that lack critical resources — including doctors — even in ordinary times.Wyoming, which did not have 1,000 total cases until June, recently added more than 1,000 in a single week. Reports of new infections have recently reached record levels in Alaska, Colorado and Idaho. And Montana, where more than half of the state’s cases have been announced since August, is averaging more than 500 cases per day.In Cascade County, more than furosemide mg 300 inmates and staff members have been infected in a facility meant to hold 365 people, the county’s first major outbreak in a region where the virus is suddenly surging.The county seat, Great Falls, is seeing its worst case numbers yet.

The local hospital and its 27-bed Covid-19 unit is at capacity. The county health department is racing to hire new contact tracers. And Mr furosemide mg. Krogue, who also teaches nursing at Montana State University’s Great Falls campus, has seen attendance in his classes dwindle as students fall ill or quarantine.“I was just scared that I’m not going to be able to see it through, that I’m going to get sick,” said Paul Krogue, the jail’s medical director.Credit...Tailyr Irvine for The New York TimesOne place where the infections have spread has been local jails, which are confined, often crowded spaces.

Jails are staples of local communities and tend to have people coming and going more quickly than prisons. Jails can hold everyone from people awaiting criminal trials for months to those picked up furosemide mg for a suspended driver’s license for a few hours. With so many people filtering in and out, jails pose extra risks for the virus’s spread — not only inside facilities but in potentially feeding outbreaks in the rest of the community.Nationally, jails and prisons have seen disproportionate rates of infection and death, with a mortality rate twice as high as in the general population and an infection rate more than four times as high, according to recent data. #styln-briefing-block { font-family.

Nyt-franklin,helvetica,arial,sans-serif. Background-color. #ffffff. Color.

Calc(100% - 40px). Border-top. 5px solid #121212. Border-bottom.

2px solid #121212. Padding. 5px 0 10px 0. } @media only screen and (min-width.

600px) { #styln-briefing-block { margin. 40px auto. } } #styln-briefing-block a { color. #121212.

} #styln-briefing-block ul { margin-left. 15px. } #styln-briefing-block a.briefing-block-link { color. #121212.

Border-bottom. 1px solid #cccccc. Font-size. 0.9375rem.

Line-height. 1.375rem. } #styln-briefing-block a.briefing-block-link:hover { border-bottom. None.

} #styln-briefing-block .briefing-block-bullet::before { content. '•'. Margin-right. 7px.

Position. Relative. } #styln-briefing-block .briefing-block-bullet:not(:last-child) { margin-bottom. 0.75em.

} #styln-briefing-block .briefing-block-header-section { margin-bottom. 16px. } #styln-briefing-block .briefing-block-header { font-weight. 700.

} @media only screen and (min-width. 600px) { #styln-briefing-block .briefing-block-header { font-size. 1.25rem. Line-height.

1.5625rem. } } #styln-briefing-block .briefing-block-header a { text-decoration. None. Color.

#333. } #styln-briefing-block .briefing-block-header a::after { content. '›'. Position.

5px. } #styln-briefing-block .briefing-block-footer { font-size. 14px. Margin-top.

1px solid #e2e2e2. */ } #styln-briefing-block .briefing-block-briefinglinks a { font-weight. Bold. Margin-right.

6px. } #styln-briefing-block .briefing-block-footer a { border-bottom. 1px solid #ccc. } #styln-briefing-block .briefing-block-footer a:hover { border-bottom.

1px solid transparent. } #styln-briefing-block .briefing-block-header { border-bottom. None. } #styln-briefing-block .briefing-block-lb-items { display.

Grid. Grid-template-columns. Auto 1fr. Grid-column-gap.

1.2. } #styln-briefing-block .briefing-block-update-time a { color. #999. Font-size.

12px. } #styln-briefing-block .briefing-block-update-time.active a { color. #D0021B. } #styln-briefing-block .briefing-block-footer-meta { display.

None. Justify-content. Space-between. Align-items.

Center. } #styln-briefing-block .briefing-block-ts { color. #D0021B. Font-size.

12px. Display. Block. } @media only screen and (min-width.

600px) { #styln-briefing-block a.briefing-block-link { font-size. 1.0625rem. Line-height. 1.5rem.

} #styln-briefing-block .briefing-block-bullet::before { content. '•'. Margin-right. 10px.

Position. Relative. } #styln-briefing-block .briefing-block-update-time a { font-size. 13px.

} } @media only screen and (min-width. 1024px) { #styln-briefing-block { width. 100%. } } Latest Updates.

The Coronavirus Outbreak 31h ago Talks on broad relief are stuck as time runs short, Pelosi says, while Senate Republicans plan narrow bills. 33h ago A frozen yogurt shop in Colorado offered maskless customers a 10 percent discount. Uproar ensued. 35h ago An infection at Pope Francis’ residence adds to concerns for his safety.

See more updates More live coverage. Markets A New York Times database has tracked clusters of at least 50 coronavirus cases in a dozen rural jails in Montana, Idaho, Utah and New Mexico during the pandemic. Among them. The Purgatory Correctional Center in Hurricane, Utah, with 166 infections.

The jail in Twin Falls, Idaho, with 279. And, in New Mexico, the Cibola County Correctional Center, which has reported 357 cases.In Cascade County, infections at the jail make up about a quarter of all known virus cases in the county. Health authorities say that the jail’s outbreak, which began in mid-August, was not believed to be the main cause of the community’s recent surge, but that it had led to some cases. In the past two months, Mr.

Krogue said, the jail released 29 people who were considered actively infected.Infections at the jail make up about a quarter of Cascade County’s known virus cases.Credit...Tailyr Irvine for The New York TimesGreat Falls, home to about 58,000 residents, is in the less mountainous part of Montana, with the Missouri River flowing through and a large oil refinery on its banks. The Cascade County Detention Center sits along a highway at the edge of town. Drive five miles in any direction and you are surrounded by wide-open plains.Montana requires that masks be worn inside businesses and indoor public spaces, and many people in Great Falls wear them when walking around downtown’s Central Avenue, where shops and cafes are still recovering from shutting down in the spring. Others go without masks, citing the open space and lack of crowds.Bob Kelly, the mayor, said people had not been overly worried about how the jail outbreak might affect the rest of town when it started.“I think that by the very definition of a jail, hopefully, the disease will be incarcerated, as well as the patients,” he said.

€œIs there concern?. Sure, there’s concern. But is there overreaction?. No.”The mayor of Great Falls said that residents had considered the jail’s outbreak a distant concern at first.Credit...Tailyr Irvine for The New York TimesSome residents’ nonchalance about the risks of the virus, said Mr.

Krogue, the jail’s medical director, can be traced to a spring and early summer when almost no one in Cascade County knew anyone who had been sickened.“We benefited from that early on,” he said. €œBut in some ways, I think it did us a disservice, too, because it also created a certain level of complacency.”That has quickly shifted now, he said, as cases have spiked.The number of active cases known to county officials on any given day has risen sharply to about 600, according to Trisha Gardner, Cascade County’s health officer. The county has seen 1,261 cases and six deaths during the pandemic, a Times database shows. Some of the cases have been tied to the jail outbreak, she said, and others have been connected to bars and restaurants.

Even figuring out what has led to some cases has been complex, she said, as residents have been reluctant to cooperate with contact tracers.“Our hospitals are at capacity, our public health system is at capacity,” she said. €œIt’s not sustainable at this rate.”When the outbreak at the jail began, social distancing was impossible, the authorities said. Three inmates shared cells designed for two. At night, men slept on thin blue pads in every available space.

On the floor in the day room, in shower stalls, in stairwells, in hallways outside of cells.Inmates did not receive masks until August, and jail officials said many have refused to wear them.In interviews with more than a dozen inmates and their family members, inmates described the jail during the outbreak as chaotic and unsanitary. They said their pleas for help often went unanswered by nurses and guards.Newly arriving inmates were not always quarantined from one another before their test results were known because of a lack of space, inmates and jail officials said.Owen Hawley, 30, said every inmate in his living area of 38 men had tested positive for the virus. He said he had been unable to eat for three days, had intensive body aches and suffered from a headache so powerful it felt as if it was “behind my eyes.”“After the fourth day of like, not eating and stuff, I just shut off, you know?. € he said.A jail area set aside for quarantining new inmates.Credit...Tailyr Irvine for The New York TimesAt one point, Mr.

Hawley said, he and other prisoners protested the way the virus was being handled by refusing to leave their living areas and by blocking new inmates from entering. Everyone was ultimately tested, Mr. Hawley said, and each prisoner was given a disposable mask.Sierra Jasmine Wells, 25, another inmate, said women in her dormitory had grown ill, one after the next.“Everyone around me was getting sick and it was tough on me,” she said. €œBy then, I had already accepted the fact that I was going to get sick.”When she became infected, she said, she was given cough syrup and Tylenol.“I kind of was just left alone to deal with it,” she said.Jesse Slaughter, the county sheriff who oversees the jail, said that the jail’s medical staff was doing everything it could, and that he had been seeking health care assistance from other counties.

Officials defended their handling of the outbreak, noting that all inmates received standard medications including Tylenol twice a day and were taken to area hospitals when they needed added care. Seven inmates, as well as some staff members, were hospitalized. No one from the jail has died from the virus, officials said.Sheriff Jesse Slaughter, who oversees the jail, said he had been seeking health care assistance from other counties.Credit...Tailyr Irvine for The New York TimesMr. Krogue said that since the start of the outbreak he had been working up to 16 hours each day and sleeping in his basement, away from his wife and children.

He remains healthy but says he fears bringing the virus home. The virus has slowed some in the jail, and officials have moved some inmates to other facilities, but other prisons and jails in the state are now seeing outbreaks.“You can start to see what some of these other places experienced much earlier on, and we just didn’t have that experience, but it’s certainly happening now,” Mr. Krogue said. €œIt’s just real in a way that it wasn’t.”Lucy Tompkins reported from Great Falls, Maura Turcotte from Chicago and Libby Seline from Lincoln, Neb.

Reporting was contributed by Izzy Colón from Columbia, Mo., Brendon Derr from Phoenix, Rebecca Griesbach from Tuscaloosa, Ala., Danya Issawi and Timothy Williams from New York, Ann Hinga Klein from Des Moines, K.B. Mensah from Silver Spring, Md., and Mitch Smith from Chicago.Start Preamble Food and Drug Administration, HHS. Notice of availability. The Food and Drug Administration (FDA or Agency) is announcing the availability of FDA guidance documents related to the Coronavirus Disease 2019 (COVID-19) public health emergency (PHE).

This notice of availability (NOA) is pursuant to the process that FDA announced, in the Federal Register of March 25, 2020, for making available to the public COVID-19-related guidances. The guidances identified in this notice address issues related to the COVID-19 PHE and have been issued in accordance with the process announced in the March 25, 2020, notice. The guidances have been implemented without prior comment, but they remain subject to comment in accordance with the Agency's good guidance practices. The announcement of the guidances is published in the Federal Register on October 16, 2020.

The guidances have been implemented without prior comment, but they remain subject to comment in accordance with the Agency's good guidance practices. You may submit either electronic or written comments on Agency guidances at any time as follows. Electronic Submissions Submit electronic comments in the following way. Federal eRulemaking Portal.

Https://www.regulations.gov. Follow the instructions for submitting comments. Comments submitted electronically, including attachments, to https://www.regulations.gov will be posted to the docket unchanged. Because your comment will be made public, you are solely responsible for ensuring that your comment does not include any confidential information that you or a third party may not wish to be posted, such as medical information, your or anyone else's Social Security number, or confidential business information, such as a manufacturing process.

Please note that if you include your name, contact information, or other information that identifies you in the body of your comments, that information will be posted on https://www.regulations.gov. If you want to submit a comment with confidential information that you do not wish to be made available to the public, submit the comment as a written/paper submission and in the manner detailed (see “Written/Paper Submissions” and “Instructions”). Written/Paper Submissions Submit written/paper submissions as follows. Mail/Hand Delivery/Courier (for written/paper submissions).

Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852. For written/paper comments submitted to the Dockets Management Staff, FDA will post your comment, as well as any attachments, except for information submitted, marked and identified, as confidential, if submitted as detailed in “Instructions.” Instructions. All submissions received must include the name of the guidance document that the comments address and the docket number for the guidance (see table 1).

Received comments will be placed in the docket(s) and, except for those submitted as “Confidential Submissions,” publicly viewable at https://www.regulations.gov or at the Dockets Management Staff between 9 a.m. And 4 p.m., Monday through Friday, 240-402-7500. Confidential Submissions—To submit a comment with confidential information that you do not wish to be made publicly available, submit your comments only as a written/paper submission. You should submit two copies total.

One copy will include the information you claim to be confidential with a heading or cover note that states “THIS DOCUMENT CONTAINS CONFIDENTIAL INFORMATION.” The Agency will review this copy, including the claimed confidential information, in Start Printed Page 65821its consideration of comments. The second copy, which will have the claimed confidential information redacted/blacked out, will be available for public viewing and posted on https://www.regulations.gov. Submit both copies to the Dockets Management Staff. If you do not wish your name and contact information to be made publicly available, you can provide this information on the cover sheet and not in the body of your comments and you must identify this information as “confidential.” Any information marked as “confidential” will not be disclosed except in accordance with 21 CFR 10.20 and other applicable disclosure law.

For more information about FDA's posting of comments to public dockets, see 80 FR 56469, September 18, 2015, or access the information at. Https://www.govinfo.gov/​content/​pkg/​FR-2015-09-18/​pdf/​2015-23389.pdf. Docket. For access to the docket to read background documents or the electronic and written/paper comments received, go to https://www.regulations.gov and insert the docket number, found in brackets in the heading of this document, into the “Search” box and follow the prompts and/or go to the Dockets Management Staff, 5630 Fishers Lane, Rm.

1061, Rockville, MD 20852, 240-402-7500. You may submit comments on any guidance at any time (see 21 CFR 10.115(g)(5)). Submit written requests for single copies of these guidances to the address noted in table 1. Send two self-addressed adhesive labels to assist that office in processing your requests.

See the SUPPLEMENTARY INFORMATION section for electronic access to the guidances. Start Further Info Kimberly Thomas, Center for Drug Evaluation and Research (CDER), Food and Drug Administration, 10903 New Hampshire Ave., Bldg. 51, Rm. 6220, Silver Spring, MD 20993-0002, 301-796-2357.

End Further Info End Preamble Start Supplemental Information I. Background On January 31, 2020, as a result of confirmed cases of COVID-19, and after consultation with public health officials as necessary, Alex M. Azar II, Secretary of Health and Human Services, pursuant to the authority under section 319 of the Public Health Service Act (42 U.S.C. 247d) (PHS Act), determined that a PHE exists and has existed since January 27, 2020, nationwide.[] On March 13, 2020, President Donald J.

Trump declared that the COVID-19 outbreak in the United States constitutes a national emergency, beginning March 1, 2020.[] In the Federal Register of March 25, 2020 (the March 25, 2020, notice) (available at https://www.govinfo.gov/​content/​pkg/​FR-2020-03-25/​pdf/​2020-06222.pdf), FDA announced procedures for making available FDA guidances related to the COVID-19 PHE. These procedures, which operate within FDA's established good guidance practices regulations, are intended to allow FDA to rapidly disseminate Agency recommendations and policies related to COVID-19 to industry, FDA staff, and other stakeholders. The March 25, 2020, notice stated that due to the need to act quickly and efficiently to respond to the COVID-19 PHE, FDA believes that prior public participation will not be feasible or appropriate before FDA implements COVID-19-related guidances. Therefore, FDA will issue COVID-19-related guidances for immediate implementation without prior public comment (see section 701(h)(1)(C) of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C.

371(h)(1)(C) and 21 CFR 10.115(g)(2) (§ 10.115(g)(2))). The guidances are available at FDA's web page entitled “COVID-19-Related Guidance Documents for Industry, FDA Staff, and Other Stakeholders” (https://www.fda.gov/​emergency-preparedness-and-response/​mcm-issues/​covid-19-related-guidance-documents-industry-fda-staff-and-other-stakeholders) and through FDA's web page entitled “Search for FDA Guidance Documents” available at https://www.fda.gov/​regulatory-information/​search-fda-guidance-documents. The March 25, 2020, notice further stated that, in general, rather than publishing a separate NOA for each COVID-19-related guidance, FDA intends to publish periodically a consolidated NOA announcing the availability of certain COVID-19-related guidances that FDA issued during the relevant period, as included in table 1. This notice announces COVID-19-related guidances that are posted on FDA's website.

II. Availability of COVID-19-Related Guidance Documents Pursuant to the process described in the March 25, 2020, notice, FDA is announcing the availability of the following COVID-19-related guidances. Table 1—Guidances Related to the COVID-19 Public Health EmergencyDocket No.CenterTitle of guidanceContact information to request single copiesFDA-2020-D-1136CDERManufacturing, Supply Chain, and Drug and Biological Product Inspections During COVID-19 Public Health Emergency Questions and Answers (August 2020)druginfo@fda.hhs.gov. Please include the docket number FDA-2020-D-1136 and complete title of the guidance in the request.FDA-2020-D-1136CDERResuming Normal Drug and Biologics Manufacturing Operations During the COVID-19 Public Health Emergency (September 2020)druginfo@fda.hhs.gov.

Please include the docket number FDA-2020-D-1136 and complete title of the guidance in the request.FDA-2020-D-1106CDERFDA Guidance on Conduct of Clinical Trials of Medical Products during COVID-19 Public Health Emergency (March 2020) (Updated September 2020)Clinicaltrialconduct-COVID19@fda.hhs.gov. Please include the docket number FDA-2020-D-1106 and complete title of the guidance in the request. Although these guidances have been implemented immediately without prior comment, FDA will consider all comments received and revise the guidances as appropriate (see § 10.115(g)(3)). These guidances are being issued consistent with FDA's good guidance practices regulation (§ 10.115).

The Start Printed Page 65822guidances represent the current thinking of FDA. They do not establish any rights for any person and are not binding on FDA or the public. You can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations. III.

Paperwork Reduction Act of 1995 CDER Guidances The guidances listed in the table below refer to previously approved FDA collections of information. Therefore, clearance by the Office of Management and Budget (OMB) under the Paperwork Reduction Act of 1995 (PRA) (44 U.S.C. 3501-3521) is not required for these guidances. However, these previously approved collections of information are subject to review by OMB under the PRA.

The collections of information in the following FDA regulations and guidances have been approved by OMB as listed in the following table. Table 2—CDER Guidances and CollectionsCOVID-19 guidance titleCFR cite referenced in COVID-19 guidanceAnother guidance title referenced in COVID-19 guidanceOMB control No(s).Guidance for Industry. Resuming Normal Drug and Biologics Manufacturing Operations During the COVID-19 Public Health Emergency21 CFR 210 and 211, 21 CFR 514.80, 21 CFR 600—Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients —Planning for the Effects of High Absenteeism to Ensure Availability of Medically Necessary Drug Products. €”Notifying FDA of a Permanent Discontinuance or Interruption in Manufacturing Under Section 506C of the FD&C Act.

€”Reporting and Mitigating Animal Drug Shortages During the COVID-19 Public Health Emergency.0910-0001, 0910-0032, 0910-0139, 0910-0338, 0910-0669, 0910-0675, 0910-0759, 0910-0806.Manufacturing, Supply Chain, and Drug and Biological Product Inspections During COVID-19 Public Health Emergency. Questions and Answers21 CFR 314.50. 314.95, 314.125, 314.127. 601.2 and 601.20—Prioritization of the Review of Original ANDAs, Amendments, and Supplements —Requests for Expedited Review of New Drug Application and Biologics License Application Prior Approval Supplements Submitted for Chemistry, Manufacturing, and Controls Changes.0910-0001, 0910-0014, 0910-0338, 0910-0045, 0910-0139, 0910-0759. —Administrative Processing of Original Biologics License Applications (BLA) and New Drug Applications (NDA). —Changes to an Approved Application for Specified Biotechnology and Specified Synthetic Biological Products. —Changes to an Approved Application.

Biological Products. —Changes to an Approved NDA or ANDA. Questions and Answers. —Changes to an Approved NDA or ANDA. —CMC Postapproval Manufacturing Changes To Be Documented in Annual Reports. —Changes to an Approved Application. Biological Products. Human Blood and Blood Components Intended for Transfusion or for Further Manufacture. —CMC Postapproval Manufacturing Changes for Specified Biological Products To Be Documented in Annual Reports. —Chemistry, Manufacturing, and Controls Changes to an Approved Application.

Certain Biological Products. —Immediate Release Solid Oral Dosage Forms. Scale-Up and Postapproval Changes. Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation. —SUPAC-IR. Questions and Answers about SUPAC-IR Guidance. —Nonsterile Semisolid Dosage Forms.

Scale-Up and Postapproval Changes. Chemistry, Manufacturing, and Controls. In Vitro Release Testing and In Vivo Bioequivalence Documentation. —SUPAC-MR. Modified Release Solid Oral Dosage Forms.

Scale-Up and Postapproval Changes. Chemistry, Manufacturing, and Controls. In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation.Start Printed Page 65823 —SUPAC. Manufacturing Equipment Addendum.

The guidance listed in the table below refers to previously approved FDA collections of information. Therefore, clearance by OMB under the PRA is not required for this guidance. However, these collections of information are subject to review by OMB under the PRA. The previously approved collections of information in the following FDA regulations and guidance have been approved by OMB as listed in the table below.

This guidance also contains a collection of information not approved under a current collection. This collection of information has been granted a PHE waiver from the PRA by the Department of Health and Human Services (HHS) on March 19, 2020, under section 319(f) of the PHS Act. Information concerning the PHE PRA waiver can be found on the HHS website at https://aspe.hhs.gov/​public-health-emergency-declaration-pra-waivers. Table 3—CDER Guidances and CollectionsCOVID-19 guidance titleCFR cite referenced in COVID-19 guidanceAnother guidance referenced in COVID-19 guidanceOMB control No(s).Collection covered by PHE PRA waiverGuidance on Conduct of Clinical Trials of Medical Products during COVID-19 Public Health Emergency (Updated September 21, 2020)21 CFR part 11, 21 CFR part 50, 21 CFR part 56, 21 CFR part 312, 21 CFR part 314, 21 CFR part 320, 21 CFR part 601, 21 CFR part 812Formal Meetings Between the FDA and Sponsors or Applicants of PDUFA Products Formal Meetings Between the FDA and Sponsors or Applicants of BsUFA Products.

Pediatric Study Plans. Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study Plans. Draft Guidance for Industry on Demonstrating Substantial Evidence of Effectiveness for Human Drug and Biological Products. Enhancing the Diversity of Clinical Trial Populations—Eligibility Criteria, Enrollment Practices, and Trial Design.

Pregnant Women. Scientific and Ethical Considerations for Inclusion in Clinical Trials. Part 11, Electronic Records. Electronic Signatures Scope and Application.0910-0001, 0910-0014, 0910-0130, 0910-0303, 0910-0338, 0910-0119, 0910-0581, 0910-0733, 0910-0078Submission by investigators of informed consent forms to third parties. Use of Electronic Records and Electronic Signatures in Clinical Investigations under 21 CFR Part 11—Questions and Answers.

Safety Reporting Requirements for INDs and BA/BE Studies. Adverse Event Reporting to IRBs—Improving Human Subject Protection.

Krogue, a nurse lowest price furosemide practitioner, recalled. €œI was just scared that I’m not going to be able to see it through, that I’m going to get sick — you just feel so exhausted and it’s just a lot.”The Mountain West, which for months avoided the worst of the pandemic, has rapidly devolved into one of the most alarming hot spots in a country that recorded its eight millionth confirmed case on Thursday, a day when more than 65,000 cases were announced nationwide, the most in a single day since July.Seventeen states, including many in the Mountain West, have added more cases in the past week than any other week of the pandemic. And the spread through sparsely populated areas of rural America has created problems in small towns that lack critical resources — including doctors — even in ordinary times.Wyoming, which did not have 1,000 total cases until June, recently added more than 1,000 in a single week. Reports of new infections have recently reached lowest price furosemide record levels in Alaska, Colorado and Idaho. And Montana, where more than half of the state’s cases have been announced since August, is averaging more than 500 cases per day.In Cascade County, more than 300 inmates and staff members have been infected in a facility meant to hold 365 people, the county’s first major outbreak in a region where the virus is suddenly surging.The county seat, Great Falls, is seeing its worst case numbers yet.

The local hospital and its 27-bed Covid-19 unit is at capacity. The county health department is racing to hire new lowest price furosemide contact tracers. And Mr. Krogue, who also teaches nursing at Montana State University’s Great Falls campus, has seen attendance in his classes dwindle as students fall ill or quarantine.“I was just scared that I’m not going to be able to see it through, that I’m going to get sick,” said Paul Krogue, the jail’s medical director.Credit...Tailyr Irvine for The New York TimesOne place where the infections have spread has been local jails, which are confined, often crowded spaces. Jails are staples of local communities and tend to have people lowest price furosemide coming and going more quickly than prisons.

Jails can hold everyone from people awaiting criminal trials for months to those picked up for a suspended driver’s license for a few hours. With so many people filtering in and out, jails pose extra risks for the virus’s spread — not only inside facilities but in potentially feeding outbreaks in the rest of the community.Nationally, jails and prisons have seen disproportionate rates of infection and death, with a mortality rate twice as high as in the general population and an infection rate more than four times as high, according to recent data. #styln-briefing-block { lowest price furosemide font-family. Nyt-franklin,helvetica,arial,sans-serif. Background-color.

#ffffff. Color. #121212. Box-sizing. Border-box.

Calc(100% - 40px). Border-top. 5px solid #121212. Border-bottom. 2px solid #121212.

Padding. 5px 0 10px 0. } @media only screen and (min-width. 600px) { #styln-briefing-block { margin. 40px auto.

} } #styln-briefing-block a { color. #121212. } #styln-briefing-block ul { margin-left. 15px. } #styln-briefing-block a.briefing-block-link { color.

#121212. Border-bottom. 1px solid #cccccc. Font-size. 0.9375rem.

Line-height. 1.375rem. } #styln-briefing-block a.briefing-block-link:hover { border-bottom. None. } #styln-briefing-block .briefing-block-bullet::before { content.

-2px. Position. Relative. } #styln-briefing-block .briefing-block-bullet:not(:last-child) { margin-bottom. 0.75em.

} #styln-briefing-block .briefing-block-header-section { margin-bottom. 16px. } #styln-briefing-block .briefing-block-header { font-weight. 700. Font-size.

1.125rem. Line-height. 1.375rem. Display. Inline-block.

Margin-bottom. 5px. } @media only screen and (min-width. 600px) { #styln-briefing-block .briefing-block-header { font-size. 1.25rem.

Line-height. 1.5625rem. } } #styln-briefing-block .briefing-block-header a { text-decoration. None. Color.

#333. } #styln-briefing-block .briefing-block-header a::after { content. '›'. Position. Relative.

Font-weight. 500. Margin-left. 5px. } #styln-briefing-block .briefing-block-footer { font-size.

14px. Margin-top. 1.25em. /* padding-top. 1.25em.

Border-top. 1px solid #e2e2e2. */ } #styln-briefing-block .briefing-block-briefinglinks a { font-weight. Bold. Margin-right.

6px. } #styln-briefing-block .briefing-block-footer a { border-bottom. 1px solid #ccc. } #styln-briefing-block .briefing-block-footer a:hover { border-bottom. 1px solid transparent.

} #styln-briefing-block .briefing-block-header { border-bottom. None. } #styln-briefing-block .briefing-block-lb-items { display. Grid. Grid-template-columns.

Auto 1fr. Grid-column-gap. 20px. Grid-row-gap. 15px.

Line-height. 1.2. } #styln-briefing-block .briefing-block-update-time a { color. #999. Font-size.

12px. } #styln-briefing-block .briefing-block-update-time.active a { color. #D0021B. } #styln-briefing-block .briefing-block-footer-meta { display. None.

Justify-content. Space-between. Align-items. Center. } #styln-briefing-block .briefing-block-ts { color.

} @media only screen and (min-width. 600px) { #styln-briefing-block a.briefing-block-link { font-size. 1.0625rem. Line-height. 1.5rem.

} #styln-briefing-block .briefing-block-bullet::before { content. '•'. Margin-right. 10px. Color.

Top. -2px. Position. Relative. } #styln-briefing-block .briefing-block-update-time a { font-size.

13px. } } @media only screen and (min-width. 1024px) { #styln-briefing-block { width. 100%. } } Latest Updates.

The Coronavirus Outbreak 31h ago Talks on broad relief are stuck as time runs short, Pelosi says, while Senate Republicans plan narrow bills. 33h ago A frozen yogurt shop in Colorado offered maskless customers a 10 percent discount. Uproar ensued. 35h ago An infection at Pope Francis’ residence adds to concerns for his safety. See more updates More live coverage.

Markets A New York Times database has tracked clusters of at least 50 coronavirus cases in a dozen rural jails in Montana, Idaho, Utah and New Mexico during the pandemic. Among them. The Purgatory Correctional Center in Hurricane, Utah, with 166 infections. The jail in Twin Falls, Idaho, with 279. And, in New Mexico, the Cibola County Correctional Center, which has reported 357 cases.In Cascade County, infections at the jail make up about a quarter of all known virus cases in the county.

Health authorities say that the jail’s outbreak, which began in mid-August, was not believed to be the main cause of the community’s recent surge, but that it had led to some cases. In the past two months, Mr. Krogue said, the jail released 29 people who were considered actively infected.Infections at the jail make up about a quarter of Cascade County’s known virus cases.Credit...Tailyr Irvine for The New York TimesGreat Falls, home to about 58,000 residents, is in the less mountainous part of Montana, with the Missouri River flowing through and a large oil refinery on its banks. The Cascade County Detention Center sits along a highway at the edge of town. Drive five miles in any direction and you are surrounded by wide-open plains.Montana requires that masks be worn inside businesses and indoor public spaces, and many people in Great Falls wear them when walking around downtown’s Central Avenue, where shops and cafes are still recovering from shutting down in the spring.

Others go without masks, citing the open space and lack of crowds.Bob Kelly, the mayor, said people had not been overly worried about how the jail outbreak might affect the rest of town when it started.“I think that by the very definition of a jail, hopefully, the disease will be incarcerated, as well as the patients,” he said. €œIs there concern?. Sure, there’s concern. But is there overreaction?. No.”The mayor of Great Falls said that residents had considered the jail’s outbreak a distant concern at first.Credit...Tailyr Irvine for The New York TimesSome residents’ nonchalance about the risks of the virus, said Mr.

Krogue, the jail’s medical director, can be traced to a spring and early summer when almost no one in Cascade County knew anyone who had been sickened.“We benefited from that early on,” he said. €œBut in some ways, I think it did us a disservice, too, because it also created a certain level of complacency.”That has quickly shifted now, he said, as cases have spiked.The number of active cases known to county officials on any given day has risen sharply to about 600, according to Trisha Gardner, Cascade County’s health officer. The county has seen 1,261 cases and six deaths during the pandemic, a Times database shows. Some of the cases have been tied to the jail outbreak, she said, and others have been connected to bars and restaurants. Even figuring out what has led to some cases has been complex, she said, as residents have been reluctant to cooperate with contact tracers.“Our hospitals are at capacity, our public health system is at capacity,” she said.

€œIt’s not sustainable at this rate.”When the outbreak at the jail began, social distancing was impossible, the authorities said. Three inmates shared cells designed for two. At night, men slept on thin blue pads in every available space. On the floor in the day room, in shower stalls, in stairwells, in hallways outside of cells.Inmates did not receive masks until August, and jail officials said many have refused to wear them.In interviews with more than a dozen inmates and their family members, inmates described the jail during the outbreak as chaotic and unsanitary. They said their pleas for help often went unanswered by nurses and guards.Newly arriving inmates were not always quarantined from one another before their test results were known because of a lack of space, inmates and jail officials said.Owen Hawley, 30, said every inmate in his living area of 38 men had tested positive for the virus.

He said he had been unable to eat for three days, had intensive body aches and suffered from a headache so powerful it felt as if it was “behind my eyes.”“After the fourth day of like, not eating and stuff, I just shut off, you know?. € he said.A jail area set aside for quarantining new inmates.Credit...Tailyr Irvine for The New York TimesAt one point, Mr. Hawley said, he and other prisoners protested the way the virus was being handled by refusing to leave their living areas and by blocking new inmates from entering. Everyone was ultimately tested, Mr. Hawley said, and each prisoner was given a disposable mask.Sierra Jasmine Wells, 25, another inmate, said women in her dormitory had grown ill, one after the next.“Everyone around me was getting sick and it was tough on me,” she said.

€œBy then, I had already accepted the fact that I was going to get sick.”When she became infected, she said, she was given cough syrup and Tylenol.“I kind of was just left alone to deal with it,” she said.Jesse Slaughter, the county sheriff who oversees the jail, said that the jail’s medical staff was doing everything it could, and that he had been seeking health care assistance from other counties. Officials defended their handling of the outbreak, noting that all inmates received standard medications including Tylenol twice a day and were taken to area hospitals when they needed added care. Seven inmates, as well as some staff members, were hospitalized. No one from the jail has died from the virus, officials said.Sheriff Jesse Slaughter, who oversees the jail, said he had been seeking health care assistance from other counties.Credit...Tailyr Irvine for The New York TimesMr. Krogue said that since the start of the outbreak he had been working up to 16 hours each day and sleeping in his basement, away from his wife and children.

He remains healthy but says he fears bringing the virus home. The virus has slowed some in the jail, and officials have moved some inmates to other facilities, but other prisons and jails in the state are now seeing outbreaks.“You can start to see what some of these other places experienced much earlier on, and we just didn’t have that experience, but it’s certainly happening now,” Mr. Krogue said. €œIt’s just real in a way that it wasn’t.”Lucy Tompkins reported from Great Falls, Maura Turcotte from Chicago and Libby Seline from Lincoln, Neb. Reporting was contributed by Izzy Colón from Columbia, Mo., Brendon Derr from Phoenix, Rebecca Griesbach from Tuscaloosa, Ala., Danya Issawi and Timothy Williams from New York, Ann Hinga Klein from Des Moines, K.B.

Mensah from Silver Spring, Md., and Mitch Smith from Chicago.Start Preamble Food and Drug Administration, HHS. Notice of availability. The Food and Drug Administration (FDA or Agency) is announcing the availability of FDA guidance documents related to the Coronavirus Disease 2019 (COVID-19) public health emergency (PHE). This notice of availability (NOA) is pursuant to the process that FDA announced, in the Federal Register of March 25, 2020, for making available to the public COVID-19-related guidances. The guidances identified in this notice address issues related to the COVID-19 PHE and have been issued in accordance with the process announced in the March 25, 2020, notice.

The guidances have been implemented without prior comment, but they remain subject to comment in accordance with the Agency's good guidance practices. The announcement of the guidances is published in the Federal Register on October 16, 2020. The guidances have been implemented without prior comment, but they remain subject to comment in accordance with the Agency's good guidance practices. You may submit either electronic or written comments on Agency guidances at any time as follows. Electronic Submissions Submit electronic comments in the following way.

Federal eRulemaking Portal. Https://www.regulations.gov. Follow the instructions for submitting comments. Comments submitted electronically, including attachments, to https://www.regulations.gov will be posted to the docket unchanged. Because your comment will be made public, you are solely responsible for ensuring that your comment does not include any confidential information that you or a third party may not wish to be posted, such as medical information, your or anyone else's Social Security number, or confidential business information, such as a manufacturing process.

Please note that if you include your name, contact information, or other information that identifies you in the body of your comments, that information will be posted on https://www.regulations.gov. If you want to submit a comment with confidential information that you do not wish to be made available to the public, submit the comment as a written/paper submission and in the manner detailed (see “Written/Paper Submissions” and “Instructions”). Written/Paper Submissions Submit written/paper submissions as follows. Mail/Hand Delivery/Courier (for written/paper submissions). Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm.

1061, Rockville, MD 20852. For written/paper comments submitted to the Dockets Management Staff, FDA will post your comment, as well as any attachments, except for information submitted, marked and identified, as confidential, if submitted as detailed in “Instructions.” Instructions. All submissions received must include the name of the guidance document that the comments address and the docket number for the guidance (see table 1). Received comments will be placed in the docket(s) and, except for those submitted as “Confidential Submissions,” publicly viewable at https://www.regulations.gov or at the Dockets Management Staff between 9 a.m. And 4 p.m., Monday through Friday, 240-402-7500.

Confidential Submissions—To submit a comment with confidential information that you do not wish to be made publicly available, submit your comments only as a written/paper submission. You should submit two copies total. One copy will include the information you claim to be confidential with a heading or cover note that states “THIS DOCUMENT CONTAINS CONFIDENTIAL INFORMATION.” The Agency will review this copy, including the claimed confidential information, in Start Printed Page 65821its consideration of comments. The second copy, which will have the claimed confidential information redacted/blacked out, will be available for public viewing and posted on https://www.regulations.gov. Submit both copies to the Dockets Management Staff.

If you do not wish your name and contact information to be made publicly available, you can provide this information on the cover sheet and not in the body of your comments and you must identify this information as “confidential.” Any information marked as “confidential” will not be disclosed except in accordance with 21 CFR 10.20 and other applicable disclosure law. For more information about FDA's posting of comments to public dockets, see 80 FR 56469, September 18, 2015, or access the information at. Https://www.govinfo.gov/​content/​pkg/​FR-2015-09-18/​pdf/​2015-23389.pdf. Docket. For access to the docket to read background documents or the electronic and written/paper comments received, go to https://www.regulations.gov and insert the docket number, found in brackets in the heading of this document, into the “Search” box and follow the prompts and/or go to the Dockets Management Staff, 5630 Fishers Lane, Rm.

1061, Rockville, MD 20852, 240-402-7500. You may submit comments on any guidance at any time (see 21 CFR 10.115(g)(5)). Submit written requests for single copies of these guidances to the address noted in table 1. Send two self-addressed adhesive labels to assist that office in processing your requests. See the SUPPLEMENTARY INFORMATION section for electronic access to the guidances.

Start Further Info Kimberly Thomas, Center for Drug Evaluation and Research (CDER), Food and Drug Administration, 10903 New Hampshire Ave., Bldg. 51, Rm. 6220, Silver Spring, MD 20993-0002, 301-796-2357. End Further Info End Preamble Start Supplemental Information I. Background On January 31, 2020, as a result of confirmed cases of COVID-19, and after consultation with public health officials as necessary, Alex M.

Azar II, Secretary of Health and Human Services, pursuant to the authority under section 319 of the Public Health Service Act (42 U.S.C. 247d) (PHS Act), determined that a PHE exists and has existed since January 27, 2020, nationwide.[] On March 13, 2020, President Donald J. Trump declared that the COVID-19 outbreak in the United States constitutes a national emergency, beginning March 1, 2020.[] In the Federal Register of March 25, 2020 (the March 25, 2020, notice) (available at https://www.govinfo.gov/​content/​pkg/​FR-2020-03-25/​pdf/​2020-06222.pdf), FDA announced procedures for making available FDA guidances related to the COVID-19 PHE. These procedures, which operate within FDA's established good guidance practices regulations, are intended to allow FDA to rapidly disseminate Agency recommendations and policies related to COVID-19 to industry, FDA staff, and other stakeholders. The March 25, 2020, notice stated that due to the need to act quickly and efficiently to respond to the COVID-19 PHE, FDA believes that prior public participation will not be feasible or appropriate before FDA implements COVID-19-related guidances.

Therefore, FDA will issue COVID-19-related guidances for immediate implementation without prior public comment (see section 701(h)(1)(C) of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C. 371(h)(1)(C) and 21 CFR 10.115(g)(2) (§ 10.115(g)(2))). The guidances are available at FDA's web page entitled “COVID-19-Related Guidance Documents for Industry, FDA Staff, and Other Stakeholders” (https://www.fda.gov/​emergency-preparedness-and-response/​mcm-issues/​covid-19-related-guidance-documents-industry-fda-staff-and-other-stakeholders) and through FDA's web page entitled “Search for FDA Guidance Documents” available at https://www.fda.gov/​regulatory-information/​search-fda-guidance-documents. The March 25, 2020, notice further stated that, in general, rather than publishing a separate NOA for each COVID-19-related guidance, FDA intends to publish periodically a consolidated NOA announcing the availability of certain COVID-19-related guidances that FDA issued during the relevant period, as included in table 1. This notice announces COVID-19-related guidances that are posted on FDA's website.

II. Availability of COVID-19-Related Guidance Documents Pursuant to the process described in the March 25, 2020, notice, FDA is announcing the availability of the following COVID-19-related guidances. Table 1—Guidances Related to the COVID-19 Public Health EmergencyDocket No.CenterTitle of guidanceContact information to request single copiesFDA-2020-D-1136CDERManufacturing, Supply Chain, and Drug and Biological Product Inspections During COVID-19 Public Health Emergency Questions and Answers (August 2020)druginfo@fda.hhs.gov. Please include the docket number FDA-2020-D-1136 and complete title of the guidance in the request.FDA-2020-D-1136CDERResuming Normal Drug and Biologics Manufacturing Operations During the COVID-19 Public Health Emergency (September 2020)druginfo@fda.hhs.gov. Please include the docket number FDA-2020-D-1136 and complete title of the guidance in the request.FDA-2020-D-1106CDERFDA Guidance on Conduct of Clinical Trials of Medical Products during COVID-19 Public Health Emergency (March 2020) (Updated September 2020)Clinicaltrialconduct-COVID19@fda.hhs.gov.

Please include the docket number FDA-2020-D-1106 and complete title of the guidance in the request. Although these guidances have been implemented immediately without prior comment, FDA will consider all comments received and revise the guidances as appropriate (see § 10.115(g)(3)). These guidances are being issued consistent with FDA's good guidance practices regulation (§ 10.115). The Start Printed Page 65822guidances represent the current thinking of FDA. They do not establish any rights for any person and are not binding on FDA or the public.

You can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations. III. Paperwork Reduction Act of 1995 CDER Guidances The guidances listed in the table below refer to previously approved FDA collections of information. Therefore, clearance by the Office of Management and Budget (OMB) under the Paperwork Reduction Act of 1995 (PRA) (44 U.S.C. 3501-3521) is not required for these guidances.

However, these previously approved collections of information are subject to review by OMB under the PRA. The collections of information in the following FDA regulations and guidances have been approved by OMB as listed in the following table. Table 2—CDER Guidances and CollectionsCOVID-19 guidance titleCFR cite referenced in COVID-19 guidanceAnother guidance title referenced in COVID-19 guidanceOMB control No(s).Guidance for Industry. Resuming Normal Drug and Biologics Manufacturing Operations During the COVID-19 Public Health Emergency21 CFR 210 and 211, 21 CFR 514.80, 21 CFR 600—Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients —Planning for the Effects of High Absenteeism to Ensure Availability of Medically Necessary Drug Products. €”Notifying FDA of a Permanent Discontinuance or Interruption in Manufacturing Under Section 506C of the FD&C Act.

€”Reporting and Mitigating Animal Drug Shortages During the COVID-19 Public Health Emergency.0910-0001, 0910-0032, 0910-0139, 0910-0338, 0910-0669, 0910-0675, 0910-0759, 0910-0806.Manufacturing, Supply Chain, and Drug and Biological Product Inspections During COVID-19 Public Health Emergency. Questions and Answers21 CFR 314.50. 314.95, 314.125, 314.127. 601.2 and 601.20—Prioritization of the Review of Original ANDAs, Amendments, and Supplements —Requests for Expedited Review of New Drug Application and Biologics License Application Prior Approval Supplements Submitted for Chemistry, Manufacturing, and Controls Changes.0910-0001, 0910-0014, 0910-0338, 0910-0045, 0910-0139, 0910-0759. —Administrative Processing of Original Biologics License Applications (BLA) and New Drug Applications (NDA). —Changes to an Approved Application for Specified Biotechnology and Specified Synthetic Biological Products. —Changes to an Approved Application. Biological Products. —Changes to an Approved NDA or ANDA.

Questions and Answers. —Changes to an Approved NDA or ANDA. —CMC Postapproval Manufacturing Changes To Be Documented in Annual Reports. —Changes to an Approved Application. Biological Products. Human Blood and Blood Components Intended for Transfusion or for Further Manufacture. —CMC Postapproval Manufacturing Changes for Specified Biological Products To Be Documented in Annual Reports. —Chemistry, Manufacturing, and Controls Changes to an Approved Application. Certain Biological Products. —Immediate Release Solid Oral Dosage Forms. Scale-Up and Postapproval Changes.

Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation. —SUPAC-IR. Questions and Answers about SUPAC-IR Guidance. —Nonsterile Semisolid Dosage Forms. Scale-Up and Postapproval Changes. Chemistry, Manufacturing, and Controls. In Vitro Release Testing and In Vivo Bioequivalence Documentation. —SUPAC-MR.

Modified Release Solid Oral Dosage Forms. Scale-Up and Postapproval Changes. Chemistry, Manufacturing, and Controls. In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation.Start Printed Page 65823 —SUPAC. Manufacturing Equipment Addendum.

The guidance listed in the table below refers to previously approved FDA collections of information. Therefore, clearance by OMB under the PRA is not required for this guidance. However, these collections of information are subject to review by OMB under the PRA. The previously approved collections of information in the following FDA regulations and guidance have been approved by OMB as listed in the table below. This guidance also contains a collection of information not approved under a current collection.

This collection of information has been granted a PHE waiver from the PRA by the Department of Health and Human Services (HHS) on March 19, 2020, under section 319(f) of the PHS Act. Information concerning the PHE PRA waiver can be found on the HHS website at https://aspe.hhs.gov/​public-health-emergency-declaration-pra-waivers. Table 3—CDER Guidances and CollectionsCOVID-19 guidance titleCFR cite referenced in COVID-19 guidanceAnother guidance referenced in COVID-19 guidanceOMB control No(s).Collection covered by PHE PRA waiverGuidance on Conduct of Clinical Trials of Medical Products during COVID-19 Public Health Emergency (Updated September 21, 2020)21 CFR part 11, 21 CFR part 50, 21 CFR part 56, 21 CFR part 312, 21 CFR part 314, 21 CFR part 320, 21 CFR part 601, 21 CFR part 812Formal Meetings Between the FDA and Sponsors or Applicants of PDUFA Products Formal Meetings Between the FDA and Sponsors or Applicants of BsUFA Products. Pediatric Study Plans. Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study Plans.

Draft Guidance for Industry on Demonstrating Substantial Evidence of Effectiveness for Human Drug and Biological Products. Enhancing the Diversity of Clinical Trial Populations—Eligibility Criteria, Enrollment Practices, and Trial Design. Pregnant Women. Scientific and Ethical Considerations for Inclusion in Clinical Trials. Part 11, Electronic Records.

Electronic Signatures Scope and Application.0910-0001, 0910-0014, 0910-0130, 0910-0303, 0910-0338, 0910-0119, 0910-0581, 0910-0733, 0910-0078Submission by investigators of informed consent forms to third parties. Use of Electronic Records and Electronic Signatures in Clinical Investigations under 21 CFR Part 11—Questions and Answers. Safety Reporting Requirements for INDs and BA/BE Studies.

Furosemide interventions

Scott Gottlieb, former Commissioner of the FDAAdam Jeffery | furosemide interventions CNBCDr. Scott Gottlieb, former FDA chief under President Donald Trump, said on Sunday that the new guidance from the Centers for Disease Control and Prevention to not test asymptomatic people for Covid-19 was "unfortunate" because those people could be at high risk of contracting the infection. "We should be testing those people to make sure they haven't become infected and aren't asymptomatic carriers because we know that they can spread the infection," Gottlieb said in an interview on CBS' "Face the Nation." "They're less likely to spread the infection, but they can still spread the infection."Earlier this month, the CDC quietly revised its guidance on coronavirus testing and dropped its previous recommendation to test everyone who has come into close contact with an infected person, even those who don't have symptoms.The move drew immediate criticism from medical groups and allegations of furosemide interventions political motivation.

Two federal health officials reportedly said the CDC was pressured into changing the guidance by top officials at the White House and Department of Health and Human Services.Medical experts and lawmakers say that early and widespread testing of people without symptoms can help mitigate the spread of the virus. Gottlieb said furosemide interventions that one reason for the CDC's decision could be that businesses were requiring people to test negative for the virus before they can return to work. He said he doesn't think the new guidance will likely be followed by states.

"If that's the case and that was a concern, there were more targeted ways to address that and speak to that problem, as opposed to making this very broad, sweeping change in the recommendations, which I think could be misinterpreted by the general public and certainly furosemide interventions by public health agencies within states," Gottlieb said. "And so I don't think this changed guidance is likely to be followed by many states." "I think it's prudent that we test people who might be at high risk of contracting the infection," Gottlieb added. — CNBC's Will Feuer contributed reporting.

Scott Gottlieb, former Commissioner what are furosemide used for of lowest price furosemide the FDAAdam Jeffery | CNBCDr. Scott Gottlieb, former FDA chief under President Donald Trump, said on Sunday that the new guidance from the Centers for Disease Control and Prevention to not test asymptomatic people for Covid-19 was "unfortunate" because those people could be at high risk of contracting the infection. "We should be testing those people to make sure they haven't become infected and aren't asymptomatic carriers because we know that they can spread the infection," Gottlieb said in an interview on CBS' "Face the Nation." "They're less likely to spread the infection, but they can still spread the infection."Earlier this month, the CDC quietly revised its guidance on coronavirus testing and dropped its previous recommendation to test everyone who has come into close contact with lowest price furosemide an infected person, even those who don't have symptoms.The move drew immediate criticism from medical groups and allegations of political motivation.

Two federal health officials reportedly said the CDC was pressured into changing the guidance by top officials at the White House and Department of Health and Human Services.Medical experts and lawmakers say that early and widespread testing of people without symptoms can help mitigate the spread of the virus. Gottlieb said that one reason for the CDC's decision could lowest price furosemide be that businesses were requiring people to test negative for the virus before they can return to work. He said he doesn't think the new guidance will likely be followed by states.

"If that's the case and that was a concern, there were more targeted ways to address that and speak to that problem, as opposed to making this very lowest price furosemide broad, sweeping change in the recommendations, which I think could be misinterpreted by the general public and certainly by public health agencies within states," Gottlieb said. "And so I don't think this changed guidance is likely to be followed by many states." "I think it's prudent that we test people who might be at high risk of contracting the infection," Gottlieb added. — CNBC's Will Feuer contributed reporting.