Comparison of analytical performance and economic value of two biosurveillance methods for tracking SARS-COV-2 variants of concern.

The development of biosurveillance programs with strong analytical performance and economically accessible protocols is essential for monitoring viral pathogens. Throughout the COVID-19 pandemic, whole-genome sequencing (WGS) has been the prevailing technology for SARS-CoV-2 variant of concern (VOC) detection. While WGS offers benefits, it is a lengthy process, financially and technically straining for scalable viral tracking. The aim of this study was to compare the analytical performance and economic feasibility of WGS and PCR mutation panels for distinguishing six known VOCs: Alpha (B.1.1.7 and Q.4), Gamma (P.1), Delta (B.1.617.2 and AY.4.2), and Omicron. (B.1.1.529.1). In all, 78 SARS-CoV-2-positive samples were collected from April to December 2021 at Northeastern University (Cabot Testing Site, Boston, MA, USA) for genotyping PCR and WGS analysis. MagMax Viral/Pathogen II Nucleic Acid Isolation and TaqPath COVID-19 Combo Kits were used for RNA extraction and SARS-CoV-2 confirmation. VOC discrimination was assessed using two TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assay layouts, and Ion Torrent WGS. In November 2021, the mutation panel demonstrated marked versatility by detecting the emerging Omicron variant reported by South Africa. SNP panel analysis yielded the following 78 VOC identifications: Alpha B.1.1.7 (N = 20), Alpha Q.4 (N = 3), Gamma P.1 (N = 1), Delta B.1.617.2 (N = 30), Delta AY.4.2 (N = 3), and Omicron B.1.1.529.1 (N = 20) with one undetermined (N = 1) sample. Genotyping mutation panels designated lineages in 77 of 78 samples, 46/78 were confirmed by WGS, while 32 samples failed WGS lineage assignment. RT-PCR genotyping panels offer pronounced throughput and sensitivity and provide an economically advantageous technique for SARS-CoV-2 biosurveillance.IMPORTANCEThe results presented in our manuscript demonstrate how the value of simplistic and reliable molecular assays coupled with the core scientific principle of standardization can be overlooked by the charm of more sophisticated assays and instrumentation. This effect can often be amplified during tumultuous public health events, such as the COVID-19 pandemic. By adapting standardized PCR mutation panels to detect prominently circulating SARS-CoV-2 variants, we were able to better assess the potential health impacts of rising positivity rates and transmission clusters within the Northeastern University population. While several literature publications utilizing genotyping PCR and NGS have a similar scope to ours, many investigations lack sufficiently standardized genotyping PCR and NGS bioinformatics inclusionary/exclusionary criteria for SARS-CoV-2 variant identification. Finally, the economic benefits of standardized PCR mutation panels would allow for global implementation of biosurveillance, rather than reserving biosurveillance to more economically developed nations.

Cross-sectional Ct distributions from qPCR tests can provide an early warning signal for the spread of COVID-19 in communities.

Background: SARS-CoV-2 PCR testing data has been widely used for COVID-19 surveillance. Existing COVID-19 forecasting models mainly rely on case counts obtained from qPCR results, even though the binary PCR results provide a limited picture of the pandemic trajectory. Most forecasting models have failed to accurately predict the COVID-19 waves before they occur. Recently a model utilizing cross-sectional population cycle threshold (Ct-the number of cycles required for the fluorescent signal to cross the background threshold) values obtained from PCR tests (Ct-based model) was developed to overcome the limitations of using only binary PCR results. In this study, we aimed to improve on COVID-19 forecasting models using features derived from the Ct-based model, to detect epidemic waves earlier than case-based trajectories. Methods: PCR data was collected weekly at Northeastern University (NU) between August 2020 and January 2022. Campus and county epidemic trajectories were generated from case counts. A novel forecasting approach was developed by enhancing a recent deep learning model with Ct-based features and applied in Suffolk County and NU campus. For this, cross-sectional Ct values from PCR data were used to generate Ct-based epidemic trajectories, including effective reproductive rate (Rt) and incidence. The improvement in forecasting performance was compared using absolute errors and residual squared errors with respect to actual observed cases at the 7-day and 14-day forecasting horizons. The model was also tested prospectively over the period January 2022 to April 2022. Results: Rt curves estimated from the Ct-based model indicated epidemic waves 12 to 14 days earlier than Rt curves from NU campus and Suffolk County cases, with a correlation of 0.57. Enhancing the forecasting models with Ct-based information significantly decreased absolute error (decrease of 49.4 and 221.5 for the 7 and 14-day forecasting horizons) and residual squared error (40.6 and 217.1 for the 7 and 14-day forecasting horizons) compared to the original model without Ct features. Conclusion: Ct-based epidemic trajectories can herald an earlier signal for impending epidemic waves in the community and forecast transmission peaks. Moreover, COVID-19 forecasting models can be enhanced using these Ct features to improve their forecasting accuracy. In this study, we make the case that public health agencies should publish Ct values along with the binary positive/negative PCR results. Early and accurate forecasting of epidemic waves can inform public health policies and countermeasures which can mitigate spread.

Expanded PCR Panel Testing for Identification of Respiratory Pathogens and Coinfections in Influenza-like Illness.

While COVID-19 has dominated Influenza-like illness (ILI) over the past few years, there are many other pathogens responsible for ILI. It is not uncommon to have coinfections with multiple pathogens in patients with ILI. The goal of this study was to identify the different organisms in symptomatic patients presenting with ILI using two different high throughput multiplex real time PCR platforms. Specimens were collected from 381 subjects presenting with ILI symptoms. All samples (nasal and nasopharyngeal swabs) were simultaneously tested on two expanded panel PCR platforms: Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, OpenArray™ plate (OA) (32 viral and bacterial targets); and Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, TaqMan™ Array card (TAC) (41 viral, fungal, and bacterial targets). Results were analyzed for concordance between the platforms and for identification of organisms responsible for the clinical presentation including possible coinfections. Very good agreement was observed between the two PCR platforms with 100% agreement for 12 viral and 3 bacterial pathogens. Of 381 specimens, approximately 58% of the samples showed the presence of at least one organism with an important incidence of co-infections (~36-40% of positive samples tested positive for two and more organisms). S. aureus was the most prevalent detected pathogen (~30%) followed by SARS-CoV-2 (~25%), Rhinovirus (~15%) and HHV6 (~10%). Co-infections between viruses and bacteria were the most common (~69%), followed by viral-viral (~23%) and bacterial-bacterial (~7%) co-infections. These results showed that coinfections are common in RTIs suggesting that syndromic panel based multiplex PCR tests could enable the identification of pathogens contributing to coinfections, help guide patient management thereby improving clinical outcomes and supporting antimicrobial stewardship.

One assay to test them all: Multiplex assays for expansion of respiratory virus surveillance.

Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Clinical performance of XpertXpress® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1 to 2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97 and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhance diagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

ONE ASSAY TO TEST THEM ALL: COMPARING MULTIPLEX ASSAYS FOR EXPANSION OF RESPIRATORY VIRUS SURVEILLANCE.

Background: Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Methods: Clinical performance of XpertXpress ® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. Results: The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1-2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97% and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. Conclusion: TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhancediagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections. IMPORTANCE: Viral respiratory infections represent a major burden globally, weighed down by the COVID-19 pandemic, and threatened by spillover of novel zoonotic influenza viruses. Since respiratory infections share clinical presentations, identification of the causing agent for patient care and public health measures requires laboratory testing for several pathogens, including potential zoonotic spillovers. Simultaneous detection of SARS-CoV-2, influenza, and RSV in a single RT-PCR accelerates time from sampling to diagnosis, preserve consumables, and streamline human resources to respond to other endemic or emerging pathogens. Multiplex assays have the potential to sustain and even expand surveillance systems, can utilize capacity/capability developed during the COVID-19 pandemic worldwide, thereby strengthening epidemic/pandemic preparedness, prevention, and response.

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from NPS. Saliva and NPS samples were collected from symptomatic and asymptomatic individuals (N = 615). Saliva samples were tested for SARS-CoV-2 using the TaqPath COVID-19 Fast PCR Combo kit 2.0 and the SDB-PCR, while NPS samples were tested by RT-PCR in RNA extracts according to the Irish national testing system. TaqPath COVID-19 Fast PCR Combo kit 2.0 detected SARS-CoV-2 in 52 saliva samples, of which 51 were also positive with the SDB-PCR. Compared to the NPS "gold standard" biospecimen method, 49 samples displayed concordant results, while three samples (35

SARS-CoV-2 variants of concern surveillance including Omicron using RT-PCR-based genotyping offers comparable performance to whole genome sequencing.

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.

Performance of the TaqMan COVID-19 Pooling Kit for detection of SARS-CoV-2 in asymptomatic and symptomatic populations.

Clinical evidence for asymptomatic cases of coronavirus disease (COVID-19) has reinforced the significance of effective surveillance testing programs. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays are considered the 'gold standard' for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. However, the labor and resource requirements can be prohibitive with respect to large testing volumes associated with the pandemic. Pooled testing algorithms may serve to increase testing capacity with more efficient resource utilization. Due to the lack of carefully curated cohorts, there is limited evidence for the applicability of RT-PCR pooling in asymptomatic COVID-19 cases. In this study, we compared the analytical sensitivity of the TaqMan™ SARS-CoV-2 Pooling Assay to detect one positive sample in a pool of five anterior nares swabs in symptomatic and asymptomatic cohorts at an institute of higher education. Positive pools were deconvoluted and each individual sample was retested using the TaqPath™ COVID-19 Combo Kit. Both assays target the open reading frame (ORF) 1ab, nucleocapsid (N), and spike (S) gene of the strain that originated in Wuhan, Hubei, China. Qualitative results demonstrated absolute agreement between pooled and deconvoluted samples in both cohorts. Independent t-test performed on Ct shifts supported an insignificant difference between cohorts with p-values of 0.306 (Orf1ab), 0.147 (N), and 0.052 (S). All negative pools were correctly reported as negative. Pooled PCR testing up to five samples is a valid method for surveillance testing of students and staff in a university setting, especially when the prevalence is expected to be low.

A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

The GLP-1 Receptor Agonist Liraglutide Increases Myocardial Glucose Oxidation Rates via Indirect Mechanisms and Mitigates Experimental Diabetic Cardiomyopathy.

BACKGROUND: Type 2 diabetes (T2D) increases risk for cardiovascular disease. Of interest, liraglutide, a therapy for T2D that activates the glucagon-like peptide-1 receptor to augment insulin secretion, reduces cardiovascular-related death in people with T2D, though it remains unknown how liraglutide produces these actions. Notably, the glucagon-like peptide-1 receptor is not expressed in ventricular cardiac myocytes, making it likely that ventricular myocardium-independent actions are involved. We hypothesized that augmented insulin secretion may explain how liraglutide indirectly mediates cardioprotection, which thereby increases myocardial glucose oxidation. METHODS: C57BL/6J male mice were fed either a low-fat diet (lean) or were subjected to experimental T2D and treated with either saline or liraglutide 3× over a 24-hour period. Mice were subsequently euthanized and had their hearts perfused in the working mode to assess energy metabolism. A separate cohort of mice with T2D were treated with either vehicle control or liraglutide for 2 weeks for the assessment of cardiac function via ultrasound echocardiography. RESULTS: Treatment of lean mice with liraglutide increased myocardial glucose oxidation without affecting glycolysis. Conversely, direct treatment of the isolated working heart with liraglutide had no effect on glucose oxidation. These findings were recapitulated in mice with T2D and associated with increased circulating insulin levels. Furthermore, liraglutide treatment alleviated diastolic dysfunction in mice with T2D, which was associated with enhanced pyruvate dehydrogenase activity, the rate-limiting enzyme of glucose oxidation. CONCLUSIONS: Our data demonstrate that liraglutide augments myocardial glucose oxidation via indirect mechanisms, which may contribute to how liraglutide improves cardiovascular outcomes in people with T2D.

Prevalence of major neurological disorders in predominantly rural northwest India.

BACKGROUND: Epidemiological studies based on hospital population, geographic isolates, smaller population, and focused groups provide valuable information on the pattern of diseases, but do not reflect on the true prevalence rates or the changing trends of disease over a period of time in different communities. The present study was undertaken to determine the prevalence and pattern of various neurological disorders in Himachal Pradesh. METHODOLOGY: Study was carried out in urban and rural population of district Kangra of Himachal Pradesh. A proportional representation was given to each area in the allocation of sample size as per probability proportional to size (PPS) method using a two-phase design: 1) A screening phase and 2) a clinical evaluation phase. All subjects were screened and a subset (screen positive and 10% of screen negative) was identified for the detailed clinical evaluation after screening. A standardized screening battery (NIMHANS protocol) was used for this purpose. An individual was confirmed as a case of neurological disorder only after clinical evaluation. RESULTS: A total of 260 (out of 10,000 studied) individuals were found positive for neurological disorders yielding a crude prevalence of 2.6%. The crude prevalence for rural areas was found to be 2.28% (206/9000), whereas the crude prevalence in urban area was found to be 5.4% (54/1000). Migraine was the most common disorder. CONCLUSION: In view of the high crude prevalence of major neurological disorders, there is a need to develop capacity among healthcare professionals regarding them.

Is calorie intake the fundamental driver of noncommunicable diseases in India - A systematic review.

BACKGROUND: Nutrition epidemiology initially focused on few nutrients thought to be responsible for noncommunicable diseases (NCDs). The database of Indian Nutrition Survey is based majorly on calorie intake. OBJECTIVE: The objective was to compare the change in the average calorie intake from 1990 to 2012 with the emerging epidemic of diabetes and hypertension (HTN) in India since 1990. METHODS: A comprehensive search was made in National Library of Medicine's PubMed database and Google Scholar from March to August 2018, on the above-mentioned subjects. Reports of national surveys (National Sample Survey Office and National Nutrition Monitoring Bureau) were included for average calorie intake among different states from year 1990 onward. Region-wise search depicted by national nutrition surveys resulted in 277 and 587 abstracts on the prevalence of HTN and diabetes mellitus, respectively. There were 51 full-text articles and abstracts on the prevalence of HTN and diabetes from the above regions. RESULTS: The average calorie intake per capita per day in the four zones of country in rural areas decreased from 1990 to 2012. An increasing trend in the prevalence of diabetes from rural areas was observed from 1994 to 2012. The per capita average calorie intake per day in urban areas from 1999 through 2011 in all zones except the eastern part of country was on rise. There was no consistent trend in the prevalence of HTN in any of the zones. CONCLUSION: It is not just an increase in calories, but a trade-off between the demand for calories and the demand for healthy lifestyles determines the prevalence of NCDs.

Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction.

Obesity and type 2 diabetes (T2D) increase the risk for cardiomyopathy, which is the presence of ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. As myocardial energy metabolism is altered during obesity/T2D (increased fatty acid oxidation and decreased glucose oxidation), we hypothesized that restricting myocardial glucose oxidation in lean mice devoid of the perturbed metabolic milieu observed in obesity/T2D would produce a cardiomyopathy phenotype, characterized via diastolic dysfunction. We tested our hypothesis via producing mice with a cardiac-specific gene knockout for pyruvate dehydrogenase (PDH, gene name Pdha1), the rate-limiting enzyme for glucose oxidation. Cardiac-specific Pdha1 deficient (Pdha1Cardiac-/-) mice were generated via crossing a tamoxifen-inducible Cre expressing mouse under the control of the alpha-myosin heavy chain (αMHC-MerCreMer) promoter with a floxed Pdha1 mouse. Energy metabolism and cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Tamoxifen administration produced an ~85% reduction in PDH protein expression in Pdha1Cardiac-/- mice versus their control littermates, which resulted in a marked reduction in myocardial glucose oxidation and a corresponding increase in palmitate oxidation. This myocardial metabolism profile did not impair systolic function in Pdha1Cardiac-/- mice, which had comparable left ventricular ejection fractions and fractional shortenings as their αMHC-MerCreMer control littermates, but did produce diastolic dysfunction as seen via the reduced mitral E/A ratio. Therefore, it does appear that forced restriction of glucose oxidation in the hearts of Pdha1Cardiac-/- mice is sufficient to produce a cardiomyopathy-like phenotype, independent of the perturbed metabolic milieu observed in obesity and/or T2D.

Nuclear myosin/actin-motored contact between homologous chromosomes is initiated by ATM kinase and homology-directed repair proteins at double-strand DNA breaks to suppress chromosome rearrangements.

We provide evidence for a mechanism of DNA repair that requires nuclear myosin/actin-dependent contact between homologous chromosomes to prevent formation of chromosomal rearrangement in human cells. We recently showed that DNA double strand breaks (DSBs) induced by γ-rays or endonucleases cause ATM-dependent contact formation between homologous chromosomes at damaged sites of transcriptionally active chromatin in G0/G1-phase cells. Here, we report that the mechanism of contact generation between homologous chromosomes also requires homology-directed repair proteins, including BRCA1, RAD51 and RAD52, and nuclear myosin/actin-motors. Moreover, inhibition of ATM kinase or deficiency in nuclear actin polymerization causes carcinogenic RET/PTC chromosome rearrangements after DSBs induction in human cells. These data suggest that DSBs in transcriptionally active euchromatin in G0/G1-phase cells are repaired through a mechanism that requires contact formation between homologous chromosomes and that this mechanism is mediated by HDR proteins and nuclear myosin/actin motors.

Differential Effects of Anesthetics and Opioid Receptor Activation on Cardioprotection Elicited by Reactive Oxygen Species-Mediated Postconditioning in Sprague-Dawley Rat Hearts.

BACKGROUND: Despite an array of cardioprotective interventions identified in preclinical models of ischemia-reperfusion (IR) injury, successful clinical translation has not been achieved. This study investigated whether drugs routinely used in clinical anesthesia influence cardioprotective effectiveness by reducing effects of reactive oxygen species (ROS), upstream triggers of cardioprotective signaling. Effects of propofol, sevoflurane, or remifentanil were compared on postischemic functional recovery induced by ROS-mediated postconditioning with Intralipid. METHODS: Recovery of left ventricular (LV) work, an index of IR injury, was measured in isolated Sprague-Dawley rat hearts subjected to global ischemia (20 minutes) and reperfusion (30 minutes). Hearts were either untreated or were treated with postconditioning with Intralipid (1%, throughout reperfusion). Propofol (10 µM), sevoflurane (2 vol%), remifentanil (3 nM), or combinations thereof were administered peri-ischemically (before and during IR). The effects of anesthetics on ROS production were measured in LV cardiac fibers by Amplex Red assay under phosphorylating and nonphosphorylating conditions. RESULTS: Recovery of LV work (expressed as percentage of the preischemic value ± standard deviation) in untreated hearts was poor (20% ± 7%) and was improved by Intralipid postconditioning (58% ± 8%, P = .001). In the absence of Intralipid postconditioning, recovery of LV work was enhanced by propofol (28% ± 9%, P = .049), sevoflurane (49% ± 5%, P < .001), and remifentanil (51% ± 6%, P < .001). The benefit of Intralipid postconditioning was abolished by propofol (33% ± 10%, P < .001), but enhanced by sevoflurane (80% ± 7%, P < .001) or remifentanil (80% ± 9%, P < .001). ROS signaling in LV fibers was abolished by propofol, but unaffected by sevoflurane or remifentanil. We conclude that propofol abolishes ROS-mediated Intralipid postconditioning by acting as a ROS scavenger. Sevoflurane and remifentanil are protective per se and provide additive cardioprotection to ROS-mediated cardioprotection. CONCLUSIONS: These divergent effects of routinely used drugs in clinical anesthesia may influence the translatability of cardioprotective therapies such as Intralipid postconditioning.

Trend of Neuropsychiatric Morbidity in Sub-Himalayan Region: An Audit of Retrospective Data Mining.

BACKGROUND: There is a paradigm shift in health loss due to premature mortality and disability from neuropsychiatric disorders with major burden in low- and middle-income countries. OBJECTIVE: To study the trend of admissions with neuropsychiatric and substance-use disorders in 3 years in psychiatry and medicine wards of tertiary care hospital in rural Himachal Pradesh. METHODOLOGY: A retrospective data mining was done from records of Inpatient wards of Dr. R. P. Government Medical College, Tanda, Himachal Pradesh, for the year 2013-2015. Demographic details and diagnosis of neuropsychiatric disorders, licit and illicit drug use, and their consequences in the form of hanging and poisoning were analyzed. RESULTS: Majority of admissions were attributed to alcohol abuse which increased in 3 years significantly in the months of July-September (P = 0.02) and October-December (P = 0.00). Almost all cases of neuropsychiatric disorders and majority of poisoning (58.2%) were observed among females. The productive young and middle age group (21-40 years) was mostly affected by all cause neuropsychiatric disorders (80.9%) and presented with poisoning (66.2%). Illicit drug abuse was on increasing drift among females. CONCLUSION: Indoor admissions were attributed to alcohol use and poisoning while neuropsychiatric disorders and substance abuse were probably dealt with at outpatient level. Treatment pertaining to mental illnesses was sought in severe cases only. Data demonstrating population burden are needed urgently to address the barriers to treatment to reduce burden.

Concordance of programmed death-ligand 1 expression between primary and metastatic non-small cell lung cancer by immunohistochemistry and RNA in situ hybridization.

We investigated the concordance of programmed death-ligand 1 (PD-L1) expression between primary cancer at initial diagnosis and metastasis at recurrence in resected non-small cell lung cancer (NSCLC). PD-L1 expression was evaluated using the SP142 assay in 37 NSCLC patients with paired primary lung cancer and surgically resected metastases at recurrence. PD-L1 positivity was defined as immunohistochemistry (IHC) and also evaluated by RNA in situ hybridization (RISH). The concordance rate of PD-L1 between primaries and metastases and correlation with clinicopathological factors were analyzed. PD-L1 expression was higher in squamous cell carcinoma, wild-type EGFR, and smokers than in non-squamous carcinoma, mutant EGFR, and never smokers, respectively. PD-L1 positivity was observed in 18.9% of primaries and 21.6% of metastases. IHC demonstrated 78.4% concordance of PD-L1 positivity between primary and metastatic cancers. In 10.8% of cases, PD-L1 positivity was higher in primaries than in metastases, and vice versa in the remaining 10.8%. By PD-L1 RISH, 35.1% of primaries and 27.0% of metastases demonstrated PD-L1 positivity. There was 62.2% concordance in PD-L1 by RISH between the primaries and metastases. Our results thus highlight the clinical importance of replacing metastases with primary archival tissue, particularly when re-biopsy is difficult at recurrence.

Alterations in fatty acid metabolism and sirtuin signaling characterize early type-2 diabetic hearts of fructose-fed rats.

Despite the fact that skeletal muscle insulin resistance is the hallmark of type-2 diabetes mellitus (T2DM), inflexibility in substrate energy metabolism has been observed in other tissues such as liver, adipose tissue, and heart. In the heart, structural and functional changes ultimately lead to diabetic cardiomyopathy. However, little is known about the early biochemical changes that cause cardiac metabolic dysregulation and dysfunction. We used a dietary model of fructose-induced T2DM (10% fructose in drinking water for 6 weeks) to study cardiac fatty acid metabolism in early T2DM and related signaling events in order to better understand mechanisms of disease. In early type-2 diabetic hearts, flux through the fatty acid oxidation pathway was increased as a result of increased cellular uptake (CD36), mitochondrial uptake (CPT1B), as well as increased ß-hydroxyacyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase activities, despite reduced mitochondrial mass. Long-chain acyl-CoA dehydrogenase activity was slightly decreased, resulting in the accumulation of long-chain acylcarnitine species. Cardiac function and overall mitochondrial respiration were unaffected. However, evidence of oxidative stress and subtle changes in cardiolipin content and composition were found in early type-2 diabetic mitochondria. Finally, we observed decreased activity of SIRT1, a pivotal regulator of fatty acid metabolism, despite increased protein levels. This indicates that the heart is no longer capable of further increasing its capacity for fatty acid oxidation. Along with increased oxidative stress, this may represent one of the earliest signs of dysfunction that will ultimately lead to inflammation and remodeling in the diabetic heart.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.