Inhibitors of SARS-CoV entry--identification using an internally-controlled dual envelope pseudovirion assay.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes

The effects of mitoquinone pretreatment on doxorubicin-induced acute cardiac dysfunction

Introduction: Doxorubicin (DOX) is a widely used anti-cancer drug notorious for its irreversible cardiac toxicity. Currently, Dexrazoxane is the only FDA-approved treatment for this toxicity. However, Dexrazoxane still bears some serious adverse events, and developing new strategies to mitigate DOX-induced heart damage is critical. Our lab has shown that pretreatment of the H9c2 myoblast cells with mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, and significantly improved cells’ resiliency to DOX. This study aimed to determine if MitoQ pretreatment can preserve cardiac function against DOX-induced damage in isolated rat hearts. Objectives: The effects of DOX and MitoQ on cardiac function were evaluated in isolated rat hearts. Moreover, the benefits of MitoQ pretreatment on DOX-induced cardiac dysfunction were also assessed. Methods: Langendorff heart preparation was performed after anesthesia of male SD rats (275-325 g). Hearts were isolated and retrograde perfused with Krebs’ buffer at a constant pressure of 80 mmHg with 37 ⁰C and pH of 7.35-7.45. Cardiac parameters, including left ventricle end-systolic pressure (LVESP), left ventricle end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP=LVESP-LVEDP), maximal rate of rise of LVP (dP/dt(max)), and heart rate (HR), were measured by a pressure transducer placed in the left ventricle of the rat heart. After obtaining a stable initial cardiac function, DOX (20 µM or 25 µM) or MitoQ (0.1-0.5 or 1-2.5 µM) were infused into the heart for 60 min. to determine the individual drug\u27s effects on the cardiac function. Moreover, another set of hearts was pretreated with MitoQ (0.25-0.5 or 1-2.5 µM) for 10-15 min before giving DOX (25 µM) to evaluate if MitoQ pretreatment would mitigate DOX-induced cardiac dysfunction. Cardiac functions were recorded every 5 min. throughout the experiments. The ratio between the final and initial recordings was calculated and compared among experimental groups. Results: Acute infusion of DOX into the isolated hearts dose-dependently reduced some cardiac parameters. Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, LVDP, and dP/dt(max) to 0.39±0.05, 0.35±0.06, and 0.26±0.05 than those of lower dose DOX infusion (20 μM, n=2; 0.77±0.01, 0.75±0.01, and 0.57±0.01), respectively. DOX had no effects on LVEDP and HR. Moreover, lower doses of MitoQ (0.1-0.5 μM, n=6) only slightly reduced HR to 0.77±0.01 without affecting other parameters. By contrast, higher doses of MitoQ (1-5 μM, n=4) reduced the ratios of LVESP, LVDP, dP/dt(max), and HR to 0.72±0.12, 0.51±0.18, and 0.45±0.17 0.65±0.07, respectively. Interestingly, MitoQ pretreatment before DOX (25 µM) exhibited better cardiac function accompanied by reduced HR than DOX alone. Higher MitoQ (1-2.5 µM) pretreatment improved the ratios of cardiac LVESP, LVDP, and dP/dt(max) to 0.67±0.14, 0.65±0.16, and 0.40±0.09, which were higher than those of lower dose MitoQ (0.25-0.5 μM, n=3; 0.49±0.11, 0.44±0.11, and 0.36±0.08), respectively. Conclusion: The preliminary data suggest that infusion of DOX into the heart acutely attenuated cardiac systolic function. Higher doses of MitoQ, not lower doses, also suppressed cardiac function. MitoQ pretreatment mitigated DOX-induced heart dysfunction. Acknowledgement: The project is funded by CCDA at PCOM

The effects of Mitoquinone on simulated ischemia/reperfusion injuries in H9c2 cells

Introduction: Reperfusion to an ischemic myocardium could result in damage termed myocardial ischemia/reperfusion (I/R) injury. Mitochondrial dysfunction is a major factor in I/R injury, producing less ATP and generating more reactive oxygen species (ROS). Mitoquinone (MitoQ) is an antioxidant that highly accumulates in the mitochondria. However, the dose-response effects and underlying mechanisms of MitoQ on simulated I/R injury have not been well established. Objectives: We hypothesized that H9c2 myoblast cells would be damaged by simulated I/R. Moreover, MitoQ would attenuate myocardial injury, characterized by increased cell viability, compared to non-treated control. Methods: The H9c2 myoblast cells (less than 20 passages) were treated with or without various concentrations of MitoQ (0.005, 0.05, 0.1, 0.5, 1, 2, 5 μM) under 3 different mediums: normal (containing 4.5 g glucose and pyruvate), low glucose (containing 1 g glucose and pyruvate), and no glucose/pyruvate medium. Three different experiments were conducted on the cells. The first experiment aimed to determine if MitoQ alone exerts different effects under different medium conditions by treating the cells with MitoQ for 24 hrs in a normal incubator. The second experiment aimed to determine if MitoQ increased cell viability under simulated ischemia conditions after MitoQ pretreatment. The third experiment aimed to determine if MitoQ increased cell viability under simulated I/R conditions after MitoQ pretreatment. Cell viability was measured by absorbance at 450 nm after adding a cell counting agent. The change in cell viability was expressed as ratios relative to the untreated controls. Results: Low concentrations of MitoQ alone slightly increased cell viability in all three mediums. The maximum increased cell viability was 1.25 ± 0.07 (n=9) at 0.005 μM MitoQ in the normal medium, 1.35 ± 0.23 (n=5, p MitoQ pretreatment exerts protection to cells in simulated ischemia conditions at certain MitoQ concentrations. The maximum increased cell viability was 1.37 ± 0.3 (n=4) at 0.01 μM MitoQ in normal medium, 1.20 ± 0.13 (n=4) at 1.0 μM MitoQ in low glucose medium, and 1.45 ± 0.24 (n=3) at 0.1 μM MitoQ in no glucose medium compared to the untreated control. MitoQ effects on simulated I/R injury will be reported in the future. Discussion: Preliminary data shows the effects of MitoQ alone and MitoQ pretreatment in ischemic conditions on cell viability is influenced by different mediums and concentrations of MitoQ

Comparison of the inhibition of an OCT3 transporter inhibitor, Nilotinib, on Doxorubicin’s effects on cardiac and cancer cell lines

Introduction Doxorubicin (DOX)-induced cardiotoxicity remains a significant barrier limiting its clinical application due to a lack of effective resolution. Targeting how DOX enters cardiac and cancer cells is a promising new strategy. Research suggests that an OCT3 transporter significantly contributes to DOX entry into the heart tissue. By contrast, it expresses much lower on breast cancer cell lines. Moreover, Nilotinib (NIB) can suppress OCT3 transporter function by 80%. Therefore, exploring the impact of NIB on the DOX’s effects on cardiac and cancer cell lines by altering DOX intracellular accumulation is intriguing. Objective First, we would establish a dose-response curve of DOX and NIB alone to assess their individual effects on cell viability. Secondly, we would record the impact of NIB on DOX entry within cardiac myoblasts (H9C2) and breast cancer cells (MCF7) through OCT3 transporter antagonism to assess if NIB can exert cardioprotective effects while maintaining DOX’s anticancer effect. Methods H9C2 myoblast and MCF7 breast cancer cells were seeded in 96-well black plates. Cells were treated with only DOX or NIB to establish a dose-response curve. Moreover, NIB was combined with DOX as a cotreatment or pretreatment regimen to evaluate the impacts of NIB on DOX’s effect. Titrated combinations of NIB (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 2 µM, 5 µM) and DOX (10 µM and 40µM) were used. Bioassays were conducted after cells were treated for 24 hours. Intracellular DOX fluorescence intensity was measured at 488/590 nm by fluoroskan. Subsequently, cell viability was detected by measuring absorbance at 450 nm after adding a cell counting reagent. The data were expressed as a ratio relative to untreated or the DOX control. Results DOX dose-dependently reduced viability of H9c2 and MCF7 cells. H9c2 cell showed significantly lower cell viability at 1 µM (0.86±0.04, n=10, p\u3c0.05) and 40 µM (0.40±0.02, n=10, p\u3c0.05) when compared to those of MCF7 cells (1.07±0.05 and 0.68±0.08 for 1 µM and 40 µM, respectively, n=7). By contrast, NIB (10 nM-2 µM) only slightly increased cell viability to 1.13±0.05 (n=11) in H9c2 cells and to 1.16±0.13 (n=7) in MCF7 cells, respectively, when compared to untreated control. The highest tested dose of NIB (5 µM) showed a similar reduction of cell viability to 0.83±0.07 in H9c2 cells and to 0.81±0.10 in MCF7 cells. Furthermore, NIB cotreatment mitigated DOX-induced damages in H9c2 by increasing cell viability to 1.28±0.07 (n=5) and 1.26±0.11 (n=7) when compared to the DOX controls (10 µM and 40µM), respectively. Interestingly, NIB cotreatment enhanced DOX’s anti-cancer effects in by decreasing MCF7 cell viability to 0.66±0.10 (n=7) and 0.70±0.09 (n=6) when compared to the DOX controls (10 µM and 40µM), respectively. The intracellular DOX fluorescence data and NIB pretreatment results are still being gathered. Conclusion DOX, not NIB, dose-dependently induced H9c2 and MCF7 cell death. Moreover, DOX-induced damage was more potent in H9c2 cells than in MCF7 cells. NIB cotreatment mildly protected H9c2 cells against DOX, whereas it increased DOX’s anti-cancer effects in MCF7 cells

Effect of digital psychoeducation and peer support on the mental health of family carers supporting individuals with psychosis in England (COPe-support): a randomised clinical trial

BACKGROUND: Psychoeducation delivered face-to-face is effective in alleviating mental health morbidities in family carers of individuals with psychosis. However, research in such interventions delivered online is scarce. We evaluated the effectiveness of a digital multicomponent intervention-COPe-support-in improving carers' mental wellbeing and caregiving-related outcomes. METHODS: In this two-arm, individually randomised, superiority trial, people aged 18 years or older who provided at least weekly support in any format for a relative or close friend affected by psychosis across England were randomly assigned (1:1) to either COPe-support or a passive online information resource using an independent online system. Participants were recruited through 30 mental health UK National Health Service trusts. The study team were masked to allocation and assessment of outcomes as all data collection took place online. Participants had access to either condition for 40 weeks and were advised to spend at least half an hour per week over the initial 20 weeks to go through materials at their own pace and to allow time to integrate knowledge and skills learned into practice. It was not feasible to mask participants or the online facilitator to intervention allocation. COPe-support provided psychoeducation on psychosis-related caregiving strategies and forums with professionals and other carers, and the control intervention comprised a passive online information resource. The primary outcome at 20 weeks was mental wellbeing measured by the Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS; minimally clinically important difference [MCID] 3). This trial is registered with ISRCTN, 89563420. FINDINGS: Between March 1, 2018, and Feb 14, 2020, 407 participants were randomly assigned, with 204 allocated to COPe-support and 203 allocated to control. The participants (mean age 53·1 years, SD 13·2) were mostly female (330 [81%] of 407 participants) and White (359 [88%] of 407 participants). 346 (85%) of 407 participants provided primary endpoint data, 174 (85%) of 204 participants in the COPe-support group and 172 (85%) of 203 participants in the control group. The mean WEMWBS score at 20 weeks was 44·5 (SD 8·31) for the COPe-support group and 43·3 (9·19) for the control group. We found no evidence of a difference in wellbeing between the two groups (adjusted mean difference 0·37, 95% CI -1·14 to 1·88; p=0·63). In the COPe-support group, 106 (52%) of 204 participants met the complier definition of a minimum of two logins in separate weeks. The complier average causal effect analysis increased the difference in WEMWBS scores (adjusted difference 0·83, 95% CI -1·45 to 3·11; p=0·47), but this was lower than the MCID. There were no adverse events. INTERPRETATION: Our findings did not support the use of COPe-support over a passive online information resource. However, further research to optimise digital interventions adjunctive to face-to-face support for carers remains important. FUNDING: National Institute for Health Research

When, where and how osteoporosis-associated fractures occur: An analysis from the global longitudinal study of osteoporosis in women (GLOW)

Objective: To examine when, where and how fractures occur in postmenopausal women. Methods: We analyzed data from the Global Longitudinal Study of Osteoporosis in Women (GLOW), including women aged ≥55 years from the United States of America, Canada, Australia and seven European countries. Women completed questionnaires including fracture data at baseline and years 1, 2 and 3. Results: Among 60,393 postmenopausal women, 4122 incident fractures were reported (86% non-hip, non-vertebral [NHNV], 8% presumably clinical vertebral and 6% hip). Hip fractures were more likely to occur in spring, with little seasonal variation for NHNV or spine fractures. Hip fractures occurred equally inside or outside the home, whereas 65% of NHNV fractures occurred outside and 61% of vertebral fractures occurred inside the home. Falls preceded 68-86% of NHNV and 68-83% of hip fractures among women aged ≤64 to ≥85 years, increasing with age. About 45% of vertebral fractures were associated with falls in all age groups except those ≥85 years, when only 24% occurred after falling. Conclusion: In this multi-national cohort, fractures occurred throughout the year, with only hip fracture having a seasonal variation, with a higher proportion in spring. Hip fractures occurred equally within and outside the home, spine fractures more often in the home, and NHNV fractures outside the home. Falls were a proximate cause of most hip and NHNV fractures. Postmenopausal women at risk for fracture need counseling about reducing potentially modifiable fracture risk factors, particularly falls both inside and outside the home and during all seasons of the year. © 2013 Costa et al

Androgen receptor expression predicts breast cancer survival: the role of genetic and epigenetic events

Background: Breast cancer outcome, including response to therapy, risk of metastasis and survival, is difficult to predict using currently available methods, highlighting the urgent need for more informative biomarkers. Androgen receptor (AR) has been implicated in breast carcinogenesis however its potential to be an informative biomarker has yet to be fully explored. In this study, AR protein levels were determined in a cohort of 73 Grade III invasive breast ductal adenocarcinomas

Nuclear Kaiso Expression Is Associated with High Grade and Triple-Negative Invasive Breast Cancer

Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Estimated stroke risk, yield, and number needed to screen for atrial fibrillation detected through single time screening: a multicountry patient-level meta-analysis of 141,220 screened individuals

BackgroundThe precise age distribution and calculated stroke risk of screen-detected atrial fibrillation (AF) is not known. Therefore, it is not possible to determine the number needed to screen (NNS) to identify one treatable new AF case (NNS-Rx) (i.e., Class-1 oral anticoagulation [OAC] treatment recommendation) in each age stratum. If the NNS-Rx is known for each age stratum, precise cost-effectiveness and sensitivity simulations can be performed based on the age distribution of the population/region to be screened. Such calculations are required by national authorities and organisations responsible for health system budgets to determine the best age cutoffs for screening programs and decide whether programs of screening should be funded. Therefore, we aimed to determine the exact yield and calculated stroke-risk profile of screen-detected AF and NNS-Rx in 5-year age strata.Methods and findingsA systematic review of Medline, Pubmed, and Embase was performed (January 2007 to February 2018), and AF-SCREEN international collaboration members were contacted to identify additional studies. Twenty-four eligible studies were identified that performed a single time point screen for AF in a general ambulant population, including people ≥65 years. Authors from eligible studies were invited to collaborate and share patient-level data. Statistical analysis was performed using random effects logistic regression for AF detection rate, and Poisson regression modelling for CHA2DS2-VASc scores. Nineteen studies (14 countries from a mix of low- to middle- and high-income countries) collaborated, with 141,220 participants screened and 1,539 new AF cases. Pooled yield of screening was greater in males across all age strata. The age/sex-adjusted detection rate for screen-detected AF in ≥65-year-olds was 1.44% (95% CI, 1.13%–1.82%) and 0.41% (95% CI, 0.31%–0.53%) for &lt;65-year-olds. New AF detection rate increased progressively with age from 0.34% (&lt;60 years) to 2.73% (≥85 years). Neither the choice of screening methodology or device, the geographical region, nor the screening setting influenced the detection rate of AF. Mean CHA2DS2-VASc scores (n = 1,369) increased with age from 1.1 (&lt;60 years) to 3.9 (≥85 years); 72% of ≥65 years had ≥1 additional stroke risk factor other than age/sex. All new AF ≥75 years and 66% between 65 and 74 years had a Class-1 OAC recommendation. The NNS-Rx is 83 for ≥65 years, 926 for 60–64 years; and 1,089 for &lt;60 years. The main limitation of this study is there are insufficient data on sociodemographic variables of the populations and possible ascertainment biases to explain the variance in the samples.ConclusionsPeople with screen-detected AF are at elevated calculated stroke risk: above age 65, the majority have a Class-1 OAC recommendation for stroke prevention, and &gt;70% have ≥1 additional stroke risk factor other than age/sex. Our data, based on the largest number of screen-detected AF collected to date, show the precise relationship between yield and estimated stroke risk profile with age, and strong dependence for NNS-RX on the age distribution of the population to be screened: essential information for precise cost-effectiveness calculations