The PARTNER trial of neoadjuvant olaparib with chemotherapy in triple-negative breast cancer.

PARTNER is a prospective, phase II-III, randomized controlled clinical trial that recruited patients with triple-negative breast cancer1,2, who were germline BRCA1 and BRCA2 wild type3. Here we report the results of the trial. Patients (n = 559) were randomized on a 1:1 basis to receive neoadjuvant carboplatin-paclitaxel with or without 150 mg olaparib twice daily, on days 3 to 14, of each of four cycles (gap schedule olaparib, research arm) followed by three cycles of anthracycline-based chemotherapy before surgery. The primary end point was pathologic complete response (pCR)4, and secondary end points included event-free survival (EFS) and overall survival (OS)5. pCR was achieved in 51% of patients in the research arm and 52% in the control arm (P = 0.753). Estimated EFS at 36 months in the research and control arms was 80% and 79% (log-rank P > 0.9), respectively; OS was 90% and 87.2% (log-rank P = 0.8), respectively. In patients with pCR, estimated EFS at 36 months was 90%, and in those with non-pCR it was 70% (log-rank P < 0.001), and OS was 96% and 83% (log-rank P < 0.001), respectively. Neoadjuvant olaparib did not improve pCR rates, EFS or OS when added to carboplatin-paclitaxel and anthracycline-based chemotherapy in patients with triple-negative breast cancer who were germline BRCA1 and BRCA2 wild type. ClinicalTrials.gov ID: NCT03150576 .

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

A high-resolution daily global dataset of statistically downscaled CMIP6 models for climate impact analyses.

A large number of historical simulations and future climate projections are available from Global Climate Models, but these are typically of coarse resolution, which limits their effectiveness for assessing local scale changes in climate and attendant impacts. Here, we use a novel statistical downscaling model capable of replicating extreme events, the Bias Correction Constructed Analogues with Quantile mapping reordering (BCCAQ), to downscale daily precipitation, air-temperature, maximum and minimum temperature, wind speed, air pressure, and relative humidity from 18 GCMs from the Coupled Model Intercomparison Project Phase 6 (CMIP6). BCCAQ is calibrated using high-resolution reference datasets and showed a good performance in removing bias from GCMs and reproducing extreme events. The globally downscaled data are available at the Centre for Environmental Data Analysis ( https://doi.org/10.5285/c107618f1db34801bb88a1e927b82317 ) for the historical (1981-2014) and future (2015-2100) periods at 0.25° resolution and at daily time step across three Shared Socioeconomic Pathways (SSP2-4.5, SSP5-3.4-OS and SSP5-8.5). This new climate dataset will be useful for assessing future changes and variability in climate and for driving high-resolution impact assessment models.

Host Derivation of Sindbis Virus Influences Mammalian Type I Interferon Response to Infection.

Arboviruses are defined by their ability to replicate in both mosquito vectors and mammalian hosts. There is good evidence that arboviruses "prime" their progeny for infection of the next host, such as via differential glycosylation of their outer glycoproteins or packaging of host ribosomal subunits. We and others have previously shown that mosquito-derived viruses more efficiently infect mammalian cells than mammalian-derived viruses. These observations are consistent with arboviruses acquiring host-specific adaptations, and we hypothesized that a virus derived from either the mammalian host or mosquito vector elicits different responses when infecting the mammalian host. Here, we perform an RNA-sequencing analysis of the transcriptional response of Human Embryonic Kidney 293 (HEK-293) cells to infection with either mosquito (Aedes albopictus, C7/10)- or mammalian (Baby Hamster Kidney, BHK-21)-derived Sindbis virus (SINV). We show that the C7/10-derived virus infection leads to a more robust transcriptional response in HEK-293s compared to infection with the BHK-derived virus. Surprisingly, despite more efficient infection, we found an increase in interferon-ß (IFN-ß) and interferon-stimulated gene (ISG) transcripts in response to the C7/10-derived virus infection versus the BHK-derived virus infection. However, translation of interferon-stimulated genes was lower in HEK-293s infected with the C7/10-derived virus, starkly contrasting with the transcriptional response. This inhibition of ISG translation is reflective of a more rapid overall shut-off of host cell translation following infection with the C7/10-derived virus. Finally, we show that the C7/10-derived virus infection of HEK-293 cells leads to elevated levels of phosphorylated eukaryotic translation elongation factor-2 (eEF2), identifying a potential mechanism leading to the more rapid shut-off of host translation. We postulate that the rapid shut-off of host translation in mammalian cells infected with the mosquito-derived virus acts to counter the IFN-ß-stimulated transcriptional response.

Editorial Commentary: Understanding Injury Epidemiology Builds Trust in Baseball Players.

Although the odds are extremely low, some baseball players are fortunate to make it to the professional ranks (minor or major league), where suffering an injury is common. During the 2011 to 2019 seasons, 112,405 injuries were reported in the Major League Baseball Health and Injury Tracking System database. Compared with other professional sports, baseball players have lower rates of returning to play, take longer to return to play, and have shorter careers after shoulder arthroscopy. By understanding injury epidemiology, the treating physician will gain player trust and understand the prognosis and how to properly return the player safely back to the field and best prolong their career.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

Host-Dependent Modifications of Packaged Alphavirus Genomic RNA Influence Virus Replication in Mammalian Cells.

Alphaviruses must interact efficiently with two distinct host environments in order to replicate and transmit between vertebrate and mosquito hosts. Some host-origin-dependent differences in virus particle composition that appear to facilitate the transmission cycle are known. However, the impact of host-mediated modification of packaged viral genomic RNA on subsequent infection has not been previously investigated. Here we show that in human (HEK-293) cells, mosquito-derived Sindbis virus (SINV) replicates and spreads faster, producing a more infectious virus than its mammalian-derived counterpart. This enhanced replication is neither a result of differences in the stability nor the production of the infecting genomic RNA. Nevertheless, purified genomic RNA from mosquito-derived SINV established infection in HEK-293 cells more efficiently than that of mammalian-derived SINV, indicating that the genomic RNA itself is different between the two producing hosts and this difference is a determinant of infection. In agreement with this idea, we show that mosquito-derived SINV genomic RNA is a more active template for translation than mammalian-derived SINV genomic RNA, and we attribute this difference to host-dependent changes in modification of packaged genomic RNA as determined by LC/MS-MS. Our data support the hypothesis that among other factors, the host-dependent modification profile of the packaged vRNA is likely to play an important role in the efficiency of SINV infection and replication in mammalian cells.

Obesity and Reverse Total Shoulder Arthroplasty.

PURPOSE OF REVIEW: Rates of obesity and reverse total shoulder arthroplasty (rTSA) in the USA have both escalated with time. Obese patients experience arthritis at higher rates than normal weight patients; therefore, these numbers go hand in hand. Obesity has been correlated with health comorbidities such as anxiety, cardiovascular disease, diabetes, and metabolic syndrome as well as poorer outcomes and higher complication rates following lower extremity arthroplasty. The current review investigates these comorbidities as they relate to obese patients undergoing rTSA. RECENT FINDINGS: Functional outcomes are similar to normal weight counterparts. Although longer operative times and a large soft tissue envelope would intuitively predispose these patients to higher risk for infection or other complications, this has not been reliably demonstrated. Technical considerations and awareness of potential risks in the obese patient demographic may aid the surgeon in preoperative planning and counseling of their patient. Obese patients undergoing rTSA have been shown to have higher risks specifically for infection, revision, and medical complications; however, this has not been consistently demonstrated in the single surgeon series where, more often, no difference in these metrics has been found. Outcomes measures and satisfaction are reliably improved, even when considering superobese patients, and majority of studies find their improvements and absolute values to be in line with their normal weight counterparts. Thus, rTSA does not seem to carry the same level of adverse risk associated with lower joint arthroplasty but potential for higher risk still bears consideration when counseling obese patients. Attention to factors that may negatively affect prosthesis positioning may optimize retention rates and limit early failure.

Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropods.

Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy to reduce the replication of mosquito-borne RNA viruses such as alphaviruses. We previously demonstrated the importance of a host cytosine methyltransferase, DNMT2, in Drosophila and viral RNA as a cellular target during pathogen-blocking. Here we report a role for DNMT2 in Wolbachia-induced alphavirus inhibition in Aedes species. Expression of DNMT2 in mosquito tissues, including the salivary glands, is elevated upon virus infection. Notably, this is suppressed in Wolbachia-colonized animals, coincident with reduced virus replication and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells is proviral, increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity is dependent on its methyltransferase activity. Finally, examining the effects of Wolbachia on modifications of viral RNA by LC-MS show a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is a vital mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental alphavirus replication and transmission processes.

Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking.

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. We, therefore, sought to understand the divergent regulation, function, and evolution of these orthologs. We describe the role of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2's role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, we identify and describe potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection and find that they are restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, our findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs.

To Improve Pain and Function, Platelet-Rich Plasma Injections May Be an Alternative to Surgery for Treating Lateral Epicondylitis: A Systematic Review.

PURPOSE: To determine whether platelet-rich plasma (PRP) injection for lateral epicondylitis offers patients comparable outcomes to lateral epicondylar surgery. METHODS: Embase, Cochrane Library, and MEDLINE databases were searched using the terms lateral epicondylitis, lateral elbow pain, tennis elbow, lateral epicondylalgia, and elbow tendinopathy individually and combined with the terms platelet-rich plasma injections and lateral epicondylar surgery. We compared pain relief, function between the 2 treatment options, and identified whether PRP injection reduced the incidence of lateral epicondylar surgery. Studies must have compared PRP injections with lateral epicondylar surgery for the treatment of lateral epicondylitis; be of Level I, II, or III evidence; and be written in the English language. RESULTS: Three studies (1 Level II and 2 Level III) met inclusion criteria. Two of the studies suggested that PRP injections offer similar relief as surgery in the short and mid-term, one study reported that PRP injections and surgery had similar outcomes in pain improvement and return to work, whereas 1 study reported that surgery may be a better long-term solution. CONCLUSIONS: In comparison with lateral epicondylar surgery, PRP injections offer similar improvements in pain and function for patients suffering from lateral epicondylitis, especially in the short- and mid-term in 2 of the 3 included investigations. Therefore, PRP injections are an appropriate alternative for the treatment of lateral epicondylitis. LEVEL OF EVIDENCE: Level III, Systematic Review of Level III or greater evidence.

The Relationship Between Energy Drink Consumption, Caffeine Content, and Nutritional Knowledge Among College Students.

We sought to determine which demographic characteristics influence energy drink consumption habits and to examine whether caffeine content and knowledge of human nutrition affect college students' decisions to consume these beverages. We used an online survey to ask 265 college students, who did not participate in a varsity sport, to complete a survey consisting of demographic questions, the General Knowledge Questionnaire for adults, and questions about energy drink consumption habits. We found, overall, that 23.1% of our sample used energy drinks. When compared to non-consumers (76.9%), users had a significantly lower GPA, were older, and preferred drinks with a higher caffeine content. Users reported that they consumed these drinks because they wanted to feel more alert and they enjoyed the taste, even though they reported adverse effects such as trouble sleeping, shaking and tremors, and stomachaches. Knowledge of human nutrition did not affect users' choice to consume these drinks. Although the majority of college students do not consume energy drinks, room for improvement remains to curb the use of these caffeinated beverages amongst college students.

Interactions between capsid and viral RNA regulate Chikungunya virus translation in a host-specific manner.

Alphaviruses are positive sense, RNA viruses commonly transmitted by an arthropod vector to a mammalian or avian host. In recent years, a number of the Alphavirus members have reemerged as public health concerns. Transmission from mosquito vector to vertebrate hosts requires an understanding of the interaction between the virus and both vertebrate and insect hosts to develop rational intervention strategies. The current study uncovers a novel role for capsid protein during Chikungunya virus replication whereby the interaction with viral RNA in the E1 coding region regulates protein synthesis processes early in infection. Studies done in both the mammalian and mosquito cells indicate that interactions between viral RNA and capsid protein have functional consequences that are host species specific. Our data support a vertebrate-specific role for capsid:vRNA interaction in temporally regulating viral translation in a manner dependent on the PI3K-AKT-mTOR pathway.

Wolbachia and Virus Alter the Host Transcriptome at the Interface of Nucleotide Metabolism Pathways.

Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show that Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.IMPORTANCE Recently developed arbovirus control strategies leverage the symbiotic bacterium Wolbachia, which spreads in insect populations and blocks viruses from replicating. While this strategy has been successful, details of how this "pathogen blocking" works are limited. Here, we use a combination of virus infections, fly genetics, and transcriptomics to show that Wolbachia and virus interact at host nucleotide metabolism pathways.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

Comparative analyses of alphaviral RNA:Protein complexes reveals conserved host-pathogen interactions.

The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly(A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants.

Viral RNA is a target for Wolbachia-mediated pathogen blocking.

The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia-mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naïve mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission.

Research Pearls: Checklists and Flowcharts to Improve Research Quality.

To instill quality in published clinical research, reporting guidelines, consisting of checklists and flowcharts, were developed to protect against reporting poorly designed research, and researchers should be aware of the available instruments and their appropriate use. With the popularity of synthetic reviews, meta-analyses, and systematic reviews, there is a greater need to assess risk of bias and study quality. This review highlights the most frequently used guidelines and checklists, risk-of-bias scales, and quality rating scales that can assist researchers with improving their research and its eventual publication.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Crucial Role of Increased Arid3a at the Pre-B and Immature B Cell Stages for B1a Cell Generation.

The Lin28b+Let7- axis in fetal/neonatal development plays a role in promoting CD5+ B1a cell generation as a B-1 B cell developmental outcome. Here we identify the Let7 target, Arid3a, as a crucial molecular effector of the B-1 cell developmental program. Arid3a expression is increased at pro-B cell stage and markedly increased at pre-B and immature B cell stages in the fetal/neonatal liver B-1 development relative to that in the Lin28b-Let7+ adult bone marrow (BM) B-2 cell development. Analysis of B-lineage restricted Lin28b transgenic (Tg) mice, Arid3a knockout and Arid3a Tg mice, confirmed that increased Arid3a allows B cell generation without requiring surrogate light chain (SLC) associated pre-BCR stage, and prevents MHC class II cell expression at the pre-B and newly generated immature B cell stages, distinct from pre-BCR dependent B development with MHC class II in adult BM. Moreover, Arid3a plays a crucial role in supporting B1a cell generation. The increased Arid3a leads higher Myc and Bhlhe41, and lower Siglec-G and CD72 at the pre-B and immature B cell stages than normal adult BM, to allow BCR signaling induced B1a cell generation. Arid3a-deficiency selectively blocks the development of B1a cells, while having no detectable effect on CD5- B1b, MZ B, and FO B cell generation resembling B-2 development outcome. Conversely, enforced expression of Arid3a by transgene is sufficient to promote the development of B1a cells from adult BM. Under the environment change between birth to adult, altered BCR repertoire in increased B1a cells occurred generated from adult BM. However, crossed with B1a-restricted VH/D/J IgH knock-in mice allowed to confirm that SLC-unassociated B1a cell increase and CLL/lymphoma generation can occur in aged from Arid3a increased adult BM. These results confirmed that in fetal/neonatal normal mice, increased Arid3a at the pre-B cell and immature B cell stages is crucial for generating B1a cells together with the environment for self-ligand reactive BCR selection, B1a cell maintenance, and potential for development of CLL/Lymphoma in aged mice.