Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the ...role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of V
11 and V
12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.
Heart Failure - Quality Improvement
Sodium glucose transport inhibitors (SGLT2i) have been shown to reduce the risk of hospitalizations and Cardiovascular (CV) death in patients with heart failure ...(HF). However, residents may be under prescribing SGLT2i for patients with HF in the outpatient setting possibly due to being unaware of newest guidelines or lack of comfort with prescribing SGLT2i for HF. The aim of this resident-led quality improvement project is to increase the rate of SGLT2i prescribed in patients with HF in the UT Tyler Internal Medicine (IM) Resident Clinic by 10% from a baseline of 16% within one month.
Using a fish-bone diagram, root-cause analysis was performed for different reasons SGLT2i are under prescribed by residents. A Pick Chart was used for intervention planning which involved reminders on conference tables aimed at the preceptor and resident during report out.
The intervention was implemented for one month and data was pulled using built EMR data analysis tool after this initial run to evaluate the percentage of SGLT2i prescriptions.
Pre-intervention data was pulled and revealed 16% of patients at the IM clinic with ICD codes indicating HF diagnosis were on SGLT2i (Figure 1). Post-intervention data analysis that used the same ICD codes showed a 10.7% increase to 26.7% of HF patients on SGLT2i (Figure 2).
At the IM clinic, our goal was to improve SGLT2i prescription by 10% among the IM residents by educating and reminding residents on their indications. SGLT-2i have emerged as a staple in improving mortality and hospital readmission of patients with HF with reduced (HFrEF) and preserved ejection fraction (HFpEF) as seen in Emperor Reduced and Emperor Preserved Trials. Despite the proven benefits of SGLT-2i in HF, their use in clinical practice remains suboptimal due to lack of awareness, concerns about side effects, and high cost.
We intend to continue to assess rates of SGLT2i prescription by analyzing SGLT2i prescription rates every six months and improving current interventions further by implementing more robust signs that are updated periodically with changing guidelines. This project can be further expanded and replicated at other associated clinics and in the inpatient setting in the future.
Diagnosing a Crisis: The Conundrum of Alpha-Gal McInnis, Angie; Root, Katherine; Rizer, Carol ...
Journal for nurse practitioners,
October 2021, 2021-10-00, 20211001, Letnik:
17, Številka:
9
Journal Article
Recenzirano
The often-encountered allergic reaction of unknown etiology can be a perplexing diagnostic conundrum in any clinical environment. Alpha-gal syndrome is an allergic reaction to mammalian meat ...consumption that occurs after the bite of certain hard ticks, mites, or chiggers. This potentially lethal allergic reaction is the result of an immunoglobulin E antibody response to a certain carbohydrate found in almost all mammalian meats and meat products. Epidemiologically, alpha-gal has been observed worldwide, but in the United States the prevalence is higher in the southeastern states because of the common hard tick vector known as the lone star tick. Providers should be aware of the recent scientific information available to assist clinicians at the point of care with diagnosis and treatment.
•The presentation of alpha-gal syndrome (AGS) can present a significant diagnostic conundrum with potentially lethal consequences.•A working knowledge of appropriate differential diagnoses that include the consideration of alpha-Gal syndrome is important to providing the best evidence-based care and management of patients presenting with acute allergic reactions.•Practitioners should be knowledgeable on how to advise patients to avoid subsequent exposure to red meat and/or meat products.•Practitioners should be aware of recent scientific updates in diagnostic and testing options for AGS.
The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that ...our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome ...with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, ...exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the ...single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
Avian influenza A viruses (IAVs) pose risks to public, agricultural, and wildlife health. Bridge hosts are spillover hosts that share habitat with both maintenance hosts (e.g., mallards) and target ...hosts (e.g., poultry). We conducted a comprehensive assessment of European starlings (
Sturnus vulgaris
), a common visitor to both urban and agricultural environments, to assess whether this species might act as a potential maintenance or bridge host for IAVs. First, we experimentally inoculated starlings with a wild bird IAV to investigate susceptibility and replication kinetics. Next, we evaluated whether IAV might spill over to starlings from sharing resources with a widespread IAV reservoir host. We accomplished this using a specially designed transmission cage to simulate natural environmental transmission by exposing starlings to water shared with IAV-infected mallards (
Anas platyrhynchos
). We then conducted a contact study to assess intraspecies transmission between starlings. In the initial experimental infection study, all inoculated starlings shed viral RNA and seroconverted. All starlings in the transmission study became infected and shed RNA at similar levels. All but one of these birds seroconverted, but detectable antibodies were relatively transient, falling to negative levels in a majority of birds by 59 days post contact. None of the contact starlings in the intraspecies transmission experiment became infected. In summary, we demonstrated that starlings may have the potential to act as IAV bridge hosts if they share water with IAV-infected waterfowl. However, starlings are unlikely to act as maintenance hosts due to limited, if any, intraspecies transmission. In addition, starlings have a relatively brief antibody response which should be considered when interpreting serology from field samples. Further study is needed to evaluate the potential for transmission from starlings to poultry, a possibility enhanced by starling’s behavioral trait of forming very large flocks which can descend on poultry facilities when natural resources are scarce.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While demographic changes in short-lived species may be observed relatively quickly in response to climate changes, measuring population responses of long-lived species requires long-term studies ...that are not always available. We analyzed data from a population of threatened Agassiz’s desert tortoises (Gopherus agassizii) at a 2.59km2 study plot in the Sonoran Desert ecosystem of Joshua Tree National Park, California, USA from 1978 to 2012 to examine variation in apparent survival and demography in this long-lived species. Transect-based, mark-recapture surveys were conducted in 10 of those years to locate living and dead tortoises. Previous modeling suggested that this area would become unsuitable as tortoise habitat under a warming and drying climate scenario. Estimated adult population size declined greatly from 1996 to 2012. The population appeared to have high apparent survival from 1978 to 1996 but apparent survival decreased from 1997 to 2002, concurrent with persistent drought. The best model relating apparent survivorship of tortoises ⩾18cm over time was based on a three year moving average of estimated winter precipitation. The postures and positions of a majority of dead tortoises found in 2012 were consistent with death by dehydration and starvation. Some live and many dead tortoises found in 2012 showed signs of predation or scavenging by mammalian carnivores. Coyote (Canis latrans) scats and other evidence from the site confirmed their role as tortoise predators and scavengers. Predation rates may be exacerbated by drought if carnivores switch from preferred mammalian prey to tortoises during dry years. Climate modeling suggests that the region will be subjected to even longer duration droughts in the future and that the plot may become unsuitable for continued tortoise survival. Our results showing wide fluctuations in apparent survival and decreasing tortoise density over time may be early signals of that possible outcome.
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