Escalation of genome defense capacity enables control of an expanding meiotic driver.

From RNA interference to chromatin silencing, diverse genome defense pathways silence selfish genetic elements to safeguard genome integrity1,2. Despite their diversity, different defense pathways share a modular organization, where numerous specificity factors identify diverse targets and common effectors silence them. In the PIWI-interacting RNA (piRNA) pathway, which controls selfish elements in the metazoan germline, diverse target RNAs are first identified by complementary base pairing with piRNAs and then silenced by PIWI-clade nucleases via enzymatic cleavage1,3. Such a binary architecture allows the defense systems to be readily adaptable, where new targets can be captured via the innovation of new specificity factors4,5. Thus, our current understanding of genome defense against lineage-specific selfish genes has been largely limited to the evolution of specificity factors, while it remains poorly understood whether other types of innovations are required. Here, we describe a new type of innovation, which escalates the defense capacity of the piRNA pathway to control a recently expanded selfish gene in Drosophila melanogaster. Through an in vivo RNAi screen for repressors of Stellate-a recently evolved and expanded selfish meiotic driver6-8-we discovered a novel defense factor, Trailblazer. Trailblazer is a transcription factor that promotes the expression of two PIWI-clade nucleases, Aub and AGO3, to match Stellate in abundance. Recent innovation in the DNA-binding domain of Trailblazer enabled it to drastically elevate Aub and AGO3 expression in the D. melanogaster lineage, thereby escalating the silencing capacity of the piRNA pathway to control expanded Stellate and safeguard fertility. As copy-number expansion is a recurrent feature of diverse selfish genes across the tree of life9-12, we envision that augmenting the defense capacity to quantitatively match selfish genes is likely a repeatedly employed defense strategy in evolution.

Sense of community and mental health: a cross-sectional analysis from a household survey in Wisconsin.

BACKGROUND: In the USA, one in five adults live with a mental illness, and researchers have estimated that nearly half of the population will have a mental illness over the course of their lifetime. Research has shown significant associations between social relationships and mental health outcomes at the individual and population levels. This study aims to examine whether sense of community, a type of social capital, is associated with mental health. METHODS: In a cross-sectional analysis, multiple logistic regression models were used to examine whether sense of community was associated with symptoms of depression, anxiety and stress reported over the last week. The analysis used data from the Survey of the Health of Wisconsin collected between 2014 and 2016. A total of 1647 observations are included in the analyses. RESULTS: Compared with those who report a positive sense of community, those with a negative sense of community had a significantly higher odds of reporting depression, anxiety and stress symptoms. Socioeconomic status is negatively associated with depression and anxiety, but not with stress. Women were more likely to experience moderate, severe, or extremely severe anxiety and stress, compared with men. CONCLUSION: This study extends current understanding of health benefits of social capital and found that individuals' sense of community is associated with reduced symptoms of depression, anxiety and stress. Further research examining mechanisms to support improved sense of community and other types of social capital could benefit health equity research.

Religion, Spirituality, and Coping During the Pandemic: Perspectives of Dementia Caregivers.

Religious and spiritual (R/S) practices support individuals during difficult situations. The COVID-19 social distancing restrictions may have limited access to R/S practices for older adults with Alzheimer's disease related dementia (ADRD) and their caregivers, affecting coping and well-being. This qualitative study explored the impact of social distancing on R/S practices and coping in ADRD-caregiver dyads from the perspective of caregivers. Interviews were conducted with 11 family caregivers of older adults with ADRD residing in nursing homes (n = 4) or private homes (n = 7). Caregivers continued individual and started virtual R/S practices which improved their ability to cope. However, organized R/S practices were unavailable for those with ADRD, but they used prayer and read religious texts which noticeably improved their mood. Healthcare professionals' sharing of individual and community R/S resources available for ADRD-caregiver dyads could decrease anxiety and agitation, while improving their ability to cope with increased isolation.

Harnessing the Electronic Medical Record to Improve the Utilization of Ultrasound Screening for Abdominal Aortic Aneurysms.

PURPOSE: The US Preventive Services Task Force recommends one-time ultrasound screening to detect abdominal aortic aneurysms (AAAs) in male smokers. Despite this recommendation, AAA screening is still underutilized. The aim of this study was to determine the effectiveness of an electronic medical record (EMR) automated ordering program in increasing AAA screening at an integrated health care system. METHODS: This study was a retrospective chart review of patients who underwent ultrasound screening for AAA from January 1, 2016, to December 31, 2021, at a geographically isolated integrated health care system. An automated ordering system was implemented in a stepwise fashion within our EMR beginning in March 2019. The number of ultrasound studies and the incidence of AAA were compared between manual referral and EMR automated ordering periods. RESULTS: A total of 4,176 patients met the inclusion criteria for this study, among whom 148 aneurysms were identified. There was an increase in the average number of monthly screening ultrasound studies performed during the automated ordering period compared with the manual referral period (105 vs 16.3 studies, P < .001). The incidence of AAA was lower in the automated ordering period compared with the manual referral period (3.2% vs 5.3%, P = .013). CONCLUSIONS: An EMR automated ordering program can increase the number of screening ultrasound studies performed for AAA, which may help clinicians identify more high-risk aneurysms requiring urgent repair.

"Please Don't Forget Us": A Descriptive Qualitative Study of Caregivers of Older Adults With Alzheimer's Disease and Related Dementias During the COVID-19 Pandemic.

The current descriptive qualitative study explored the perceived impact of the coronavirus disease 2019 pandemic on sleep disturbances and nighttime agitation; the reported use of antipsychotics and other sedating medications; and the overall well-being of older adults with Alzheimer's disease and related dementias (ADRD) and their caregivers. One investigator conducted in-depth, phone interviews with caregivers of nursing home residents with ADRD (four family caregivers [FCs], three nurse practitioners [NPs]) and seven FCs of older adults with ADRD who lived with them at home. Caregivers described multiple sleep disturbances. Nighttime agitation symptoms were perceived to continue or worsen, and sedating medications and nonpharmacological interventions were required. Adverse impacts on reported well-being were significant, and impacts were grouped into emotional, social, and physical themes. Caregivers said, "Please don't forget us," and requested telehealth support for those at home and technology and human resources for nursing homes to reduce adverse impacts. [Research in Gerontological Nursing, 15(5), 217-228.].

Draft Genome Sequences of Three Antibiotic-Producing Soil Bacteria, Staphylococcus pasteuri WAM01, Peribacillus butanolivorans WAM04, and Micrococcus yunnanensis WAM06, with Growth-Inhibiting Effects against Commensal Neisseria Strains.

We report the isolation, identification, and assemblies of three antibiotic-producing soil bacteria (Staphylococcus pasteuri, Peribacillus butanolivorans, and Micrococcus yunnanensis) that inhibit the growth of Neisseria commensals in coculture. With pathogenic Neisseria strains becoming increasingly resistant to antibiotics, bioprospecting for novel antimicrobials using commensal relatives may facilitate discovery of clinically useful drugs.

Seasonal COVID-19 surge related hospital volumes and case fatality rates.

BACKGROUND: Seasonal and regional surges in COVID-19 have imposed substantial strain on healthcare systems. Whereas sharp inclines in hospital volume were accompanied by overt increases in case fatality rates during the very early phases of the pandemic, the relative impact during later phases of the pandemic are less clear. We sought to characterize how the 2020 winter surge in COVID-19 volumes impacted case fatality in an adequately-resourced health system. METHODS: We performed a retrospective cohort study of all adult diagnosed with COVID-19 in a large academic healthcare system between August 25, 2020 to May 8, 2021, using multivariable logistic regression to examine case fatality rates across 3 sequential time periods around the 2020 winter surge: pre-surge, surge, and post-surge. Subgroup analyses of patients admitted to the hospital and those receiving ICU-level care were also performed. Additionally, we used multivariable logistic regression to examine risk factors for mortality during the surge period. RESULTS: We studied 7388 patients (aged 52.8 ± 19.6 years, 48% male) who received outpatient or inpatient care for COVID-19 during the study period. Patients treated during surge (N = 6372) compared to the pre-surge (N = 536) period had 2.64 greater odds (95% CI 1.46-5.27) of mortality after adjusting for sociodemographic and clinical factors. Adjusted mortality risk returned to pre-surge levels during the post-surge period. Notably, first-encounter patient-level measures of illness severity appeared higher during surge compared to non-surge periods. CONCLUSIONS: We observed excess mortality risk during a recent winter COVID-19 surge that was not explained by conventional risk factors or easily measurable variables, although recovered rapidly in the setting of targeted facility resources. These findings point to how complex interrelations of population- and patient-level pandemic factors can profoundly augment health system strain and drive dynamic, if short-lived, changes in outcomes.

Temporal variations in the severity of COVID-19 illness by race and ethnicity.

INTRODUCTION: Early reports highlighted racial/ethnic disparities in the severity of COVID-19 seen across the USA; the extent to which these disparities have persisted over time remains unclear. Our research objective was to understand temporal trends in racial/ethnic variation in severity of COVID-19 illness presenting over time. METHODS: We conducted a retrospective cohort analysis using longitudinal data from Cedars-Sinai Medical Center, a high-volume health system in Southern California. We studied patients admitted to the hospital with COVID-19 illness from 4 March 2020 through 5 December 2020. Our primary outcome was COVID-19 severity of illness among hospitalised patients, assessed by racial/ethnic group status. We defined overall illness severity as an ordinal outcome: hospitalisation but no intensive care unit (ICU) admission; admission to the ICU but no intubation; and intubation or death. RESULTS: A total of 1584 patients with COVID-19 with available demographic and clinical data were included. Hispanic/Latinx compared with non-Hispanic white patients had higher odds of experiencing more severe illness among hospitalised patients (OR 2.28, 95% CI 1.62 to 3.22) and this disparity persisted over time. During the initial 2 months of the pandemic, non-Hispanic blacks were more likely to suffer severe illness than non-Hispanic whites (OR 2.02, 95% CI 1.07 to 3.78); this disparity improved by May, only to return later in the pandemic. CONCLUSION: In our patient sample, the severity of observed COVID-19 illness declined steadily over time, but these clinical improvements were not seen evenly across racial/ethnic groups; greater illness severity continues to be experienced among Hispanic/Latinx patients.

A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics.

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein-protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

Identification of Functional Limitations and Discharge Destination in Patients With COVID-19.

OBJECTIVES: The objectives of this study were to identify functional limitations in patients with coronavirus 2019 (COVID-19) admitted to acute care hospitals; to evaluate functional limitations by demographic, medical, and encounter characteristics; and to examine functional limitations in relation to discharge destination. DESIGN: and Setting:This is a cross-sectional, retrospective study of adult patients with COVID-19 who were discharged from 2 different types of hospitals (academic medical center and a community hospital) within 1 health care system from January 1 to April 30, 2020. PARTICIPANTS: Patients were identified from the Cedars-Sinai COVID-19 data registry who had a new-onset positive test for severe acute respiratory syndrome coronavirus 2. A total of 273 patients were identified, which included 230 patients who were discharged alive and 43 patients who died and were excluded from the study sample. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Functional limitations in patients with COVID-19 in acute care hospitals and the predictors for discharge disposition. RESULTS: A total of 230 records were analyzed including demographic, encounter, medical, and functional variables. In a propensity score-matched cohort based on age and comorbidity, 88.2% had functional physical health deficits, 72.5% had functional mental health deficits, and 17.6% experienced sensory deficits. In the matched cohort, individuals discharged to an institution experienced greater physical (62.7% vs 25.5%, P<.001) and mental health (49.0% vs 23.5%, P=.006) deficits than patients discharged home. Marital status (odds ratio, 3.17; P=.011) and physical function deficits (odds ratio, 3.63; P=.025) were associated with an increase odds ratio of discharge to an institution. CONCLUSIONS: This research highlights that functional status is a strong predictor for discharge destination to an institution for patients with COVID-19. Patients who were older, in the acute care hospital longer, and with comorbidities were more likely to be discharged to an institution. Rehabilitation is a significant aspect of the health care system for these vulnerable patients. The challenges of adjusting the role of rehabilitation providers and systems during the pandemic needs further exploration. Moreover, additional research is needed to look more closely at the many facets and timing of functional status needs, to shed light in use of interdisciplinary rehabilitation services, and to guide providers and health care systems in facilitating optimal recovery and patient outcomes.

Pre-existing traits associated with Covid-19 illness severity.

IMPORTANCE: Certain individuals, when infected by SARS-CoV-2, tend to develop the more severe forms of Covid-19 illness for reasons that remain unclear. OBJECTIVE: To determine the demographic and clinical characteristics associated with increased severity of Covid-19 infection. DESIGN: Retrospective observational study. We curated data from the electronic health record, and used multivariable logistic regression to examine the association of pre-existing traits with a Covid-19 illness severity defined by level of required care: need for hospital admission, need for intensive care, and need for intubation. SETTING: A large, multihospital healthcare system in Southern California. PARTICIPANTS: All patients with confirmed Covid-19 infection (N = 442). RESULTS: Of all patients studied, 48% required hospitalization, 17% required intensive care, and 12% required intubation. In multivariable-adjusted analyses, patients requiring a higher levels of care were more likely to be older (OR 1.5 per 10 years, P<0.001), male (OR 2.0, P = 0.001), African American (OR 2.1, P = 0.011), obese (OR 2.0, P = 0.021), with diabetes mellitus (OR 1.8, P = 0.037), and with a higher comorbidity index (OR 1.8 per SD, P<0.001). Several clinical associations were more pronounced in younger compared to older patients (Pinteraction<0.05). Of all hospitalized patients, males required higher levels of care (OR 2.5, P = 0.003) irrespective of age, race, or morbidity profile. CONCLUSIONS AND RELEVANCE: In our healthcare system, greater Covid-19 illness severity is seen in patients who are older, male, African American, obese, with diabetes, and with greater overall comorbidity burden. Certain comorbidities paradoxically augment risk to a greater extent in younger patients. In hospitalized patients, male sex is the main determinant of needing more intensive care. Further investigation is needed to understand the mechanisms underlying these findings.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain.

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

Experience With Hydroxychloroquine and Azithromycin in the Coronavirus Disease 2019 Pandemic: Implications for QT Interval Monitoring.

Background Despite a lack of clinical evidence, hydroxychloroquine and azithromycin are being administered widely to patients with verified or suspected coronavirus disease 2019 (COVID-19). Both drugs may increase risk of lethal arrhythmias associated with QT interval prolongation. Methods and Results We analyzed a case series of COVID-19-positive/suspected patients admitted between February 1, 2020, and April 4, 2020, who were treated with azithromycin, hydroxychloroquine, or a combination of both drugs. We evaluated baseline and postmedication QT interval (corrected QT interval [QTc]; Bazett) using 12-lead ECGs. Critical QTc prolongation was defined as follows: (1) maximum QTc ≥500 ms (if QRS <120 ms) or QTc ≥550 ms (if QRS ≥120 ms) and (2) QTc increase of ≥60 ms. Tisdale score and Elixhauser comorbidity index were calculated. Of 490 COVID-19-positive/suspected patients, 314 (64%) received either/both drugs and 98 (73 COVID-19 positive and 25 suspected) met study criteria (age, 62±17 years; 61% men). Azithromycin was prescribed in 28%, hydroxychloroquine in 10%, and both in 62%. Baseline mean QTc was 448±29 ms and increased to 459±36 ms (P=0.005) with medications. Significant prolongation was observed only in men (18±43 ms versus -0.2±28 ms in women; P=0.02). A total of 12% of patients reached critical QTc prolongation. Changes in QTc were highest with the combination compared with either drug, with much greater prolongation with combination versus azithromycin (17±39 ms versus 0.5±40 ms; P=0.07). No patients manifested torsades de pointes. Conclusions Overall, 12% of patients manifested critical QTc prolongation, and the combination caused greater prolongation than either drug alone. The balance between uncertain benefit and potential risk when treating COVID-19 patients should be carefully assessed.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation.

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

The role of calmodulin in regulating calcium-permeable PKD2L1 channel activity.

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators.

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers.

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.