A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity.

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays, including gene expression analysis, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. SIGNIFICANCE: Bioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc luciferase, which is small in size and displays much enhanced bioluminescence and thermal stability compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or cellular signaling reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.

Plasma neurological biomarkers as a measure of neurotoxicity in pediatric dental general anesthesia: a prospective observational feasibility study.

PURPOSE: Neurotoxicity concerns have been raised over general anesthesia and sedation medication use in children. Such concerns are largely based on animal studies, historical anesthetic agents, and assessment tools, thus warranting further investigations. Blood biomarkers in detecting neuronal inflammation and apoptosis are novel methods for detecting neuronal damage. Therefore, the aim of this feasibility study was to assess the usefulness of the levels of four plasma biomarkers in dental general anesthesia (DGA) as surrogate markers of neurotoxicity in children. The secondary aim was to compare changes in motor manipulative skills pre- and post-anesthetic exposure. METHODS: This single-center prospective observational study included 22 healthy children aged between 3 and 6 years old who underwent DGA. Subclinical neurotoxicity was measured with a panel of four plasma biomarkers: Caspase-3, neuron-specific enolase (NSE), neurofilament light chain, and S100B at three time points (1; at start, 2; end and 3; on recovery from DGA). The Skillings-Mack test was used to identify the difference in the biomarker levels at three time points. Motor manipulative score assessment, prior and two weeks after DGA was also performed. RESULTS: A total of 22 study participants (mean age = 5 ± 1 years) were included with a median DGA duration of 106 ± 28 min. A reduction in Caspase-3 levels was recorded, with pairwise comparison over three time points, reporting a statistical significance between time point 2 vs. 1 and time point 3 vs. 1. Although fluctuations in NSE levels were recorded, no significant changes were found following pairwise comparison analysis. Among other biomarkers, no significant changes over the three periods were recorded. Furthermore, no significant changes in manipulative motor scores were reported. CONCLUSION: Caspase-3 reduced significantly in the short time frames during day-care DGA; this might be due to the relatively short anesthesia duration associated with dental treatment as compared with more extensive medical-related treatments. Therefore, further studies on Caspase-3 as a potential biomarker in pediatric DGA neurotoxicity are required to further ascertain results of this study.

A continuous cuffless blood pressure measurement from optimal PPG characteristic features using machine learning algorithms.

Background and objective: Hypertension is a potentially dangerous health condition that can be detected by measuring blood pressure (BP). Blood pressure monitoring and measurement are essential for preventing and treating cardiovascular diseases. Cuff-based devices, on the other hand, are uncomfortable and prevent continuous BP measurement. Methods: In this study, a new non-invasive and cuff-less method for estimating Systolic Blood Pressure (SBP), Mean Arterial Pressure (MAP), and Diastolic Blood Pressure (DBP) has been proposed using characteristic features of photoplethysmogram (PPG) signals and nonlinear regression algorithms. PPG signals were collected from 219 participants, which were then subjected to preprocessing and feature extraction steps. Analyzing PPG and its derivative signals, a total of 46 time, frequency, and time-frequency domain features were extracted. In addition, the age and gender of each subject were also included as features. Further, correlation-based feature selection (CFS) and Relief F feature selection (ReliefF) techniques were used to select the relevant features and reduce the possibility of over-fitting the models. Finally, support vector regression (SVR), K-nearest neighbour regression (KNR), decision tree regression (DTR), and random forest regression (RFR) were established to develop the BP estimation model. Regression models were trained and evaluated on all features as well as selected features. The best regression models for SBP, MAP, and DBP estimations were selected separately. Results: The SVR model, along with the ReliefF-based feature selection algorithm, outperforms other algorithms in estimating the SBP, MAP, and DBP with the mean absolute error of 2.49, 1.62 and 1.43 mmHg, respectively. The proposed method meets the Advancement of Medical Instrumentation standard for BP estimations. Based on the British Hypertension Society standard, the results also fall within Grade A for SBP, MAP, and DBP. Conclusion: The findings show that the method can be used to estimate blood pressure non-invasively, without using a cuff or calibration, and only by utilizing the PPG signal characteristic features.

Morpho-physiological and phytohormonal changes during the induction of adventitious root development stimulated by exogenous IBA application in Magnolia biondii Pamp

Abstract Magnolia biondii Pamp is an important ornamental tree species widely grown and used as a rootstock in the propagation of different Magnolia varieties. In the current studies, anatomical, physiological and endogenous hormones were studied to check the effect of IBA 750 mg/L on the adventitious rooting and to provide theoretical and technical support for the propagation of Magnolia biondii Pamp through stem cuttings. Two thousand stem cuttings were prepared and divided into two groups i.e., IBA treated cuttings and water control. For the evaluation of antioxidant enzyme activities, and endogenous hormones levels, samples were collected on the day of planting and each 5th day and further steps were carried out in the laboratory according to the protocols and proper precautions. For the anatomical observations, samples were collected on the 13th, 15th, and 17th day for IBA treated cuttings while 21st, 23rd, and 25th day for control. Collected samples were preserved in the FAA solution and further observations were carried out in the laboratory. Anatomical observations showed that it took 13 days for the differentiation of root primordia to the appearance of young adventitious roots in IBA treated cuttings, while it took 21 days to develop primordia in the control. Antioxidant enzyme activities involved in ROS were significantly higher in the IBA treated cuttings compared to control. POD showed a peak on the 13th day before the emergence of roots in IBA treated cuttings while it showed a peak on the 21st day in the control. PPO showed a peak on the 21st day in the IBA treated cuttings while it showed a peak on the 29th day in the control. SOD showed a peak on the 17th day in IBA treated cuttings, while it showed a peak on the 25th day in the control. Exogenous application of IBA enhanced the endogenous IAA and GA3 levels compared to CK, while it reduced the levels of ABA continuously at the time of rooting and then increased gradually. Inclusively, our study suggests that IBA 750 mg/L is efficient for the rooting of Magnolia biondii Pamp cuttings, as it enhanced the process of antioxidant enzyme activities, endogenous hormones levels and reduced the time of root formation which is evident from the anatomical observations.

Resumo Magnolia biondii Pamp é uma importante espécie de árvore ornamental muito cultivada e utilizada como porta-enxerto na propagação de diferentes variedades de Magnolia. Nos estudos atuais, hormônios anatômicos, fisiológicos e endógenos foram estudados para verificar o efeito do AIB na dose de 750 mg / L no enraizamento adventício e fornecer suporte teórico e técnico para a propagação de M. biondii Pamp por meio de estacas. Duas mil estacas foram preparadas e divididas em dois grupos, ou seja, tratadas com AIB e controle de água. Para a avaliação das atividades das enzimas antioxidantes e dos níveis de hormônios endógenos, as amostras foram coletadas no dia do plantio e a cada 5 dias, enquanto as demais etapas foram realizadas em laboratório de acordo com os protocolos e os devidos cuidados. Para as observações anatômicas, as amostras foram coletadas no 13º, 15º e 17º dias para estacas tratadas com AIB e no 21º, 23º e 25º dias para o controle. As amostras coletadas foram preservadas em solução FAA, e outras observações foram realizadas em laboratório. Observações anatômicas mostraram a necessidade de 13 dias para a diferenciação dos primórdios radiculares até o aparecimento de raízes adventícias jovens em estacas tratadas com AIB e de 21 dias para o desenvolvimento dos primórdios no controle. As atividades das enzimas antioxidantes envolvidas nas ROS foram significativamente maiores nas estacas tratadas com AIB em comparação com o controle. A POD apresentou pico no 13º dia antes da emergência das raízes nas estacas tratadas com AIB, enquanto no 21º dia apresentou pico no controle. A PPO teve pico no 21º dia nas estacas tratadas com AIB e no 29º dia no controle. A SOD apresentou pico no 17º dia nas estacas tratadas com AIB e no 25º dia no controle. A aplicação exógena de AIB aumentou os níveis endógenos de IAA e GA3 em relação ao controle, enquanto reduziu os níveis de ABA continuamente no momento do enraizamento e, em seguida, aumentou gradativamente. Inclusive, nosso estudo sugere que o AIB na dose de 750 mg / L é eficiente para o enraizamento de estacas de M. biondii Pamp, visto que potencializou o processo de atividades de enzimas antioxidantes e os níveis de hormônios endógenos, além de reduzir o tempo de formação de raízes, o que fica evidente nas observações anatômicas.

Morpho-physiological and phytohormonal changes during the induction of adventitious root development stimulated by exogenous IBA application in Magnolia biondii Pamp / Alterações morfofisiológicas e fito-hormonais durante a indução do desenvolvimento radicular adventício estimulado pela aplicação exógena do AIB em Magnolia biondii Pamp

Magnolia biondii Pamp is an important ornamental tree species widely grown and used as a rootstock in the propagation of different Magnolia varieties. In the current studies, anatomical, physiological and endogenous hormones were studied to check the effect of IBA 750 mg/L on the adventitious rooting and to provide theoretical and technical support for the propagation of Magnolia biondii Pamp through stem cuttings. Two thousand stem cuttings were prepared and divided into two groups i.e., IBA treated cuttings and water control. For the evaluation of antioxidant enzyme activities, and endogenous hormones levels, samples were collected on the day of planting and each 5th day and further steps were carried out in the laboratory according to the protocols and proper precautions. For the anatomical observations, samples were collected on the 13th, 15th, and 17th day for IBA treated cuttings while 21st, 23rd, and 25th day for control. Collected samples were preserved in the FAA solution and further observations were carried out in the laboratory. Anatomical observations showed that it took 13 days for the differentiation of root primordia to the appearance of young adventitious roots in IBA treated cuttings, while it took 21 days to develop primordia in the control. Antioxidant enzyme activities involved in ROS were significantly higher in the IBA treated cuttings compared to control. POD showed a peak on the 13th day before the emergence of roots in IBA treated cuttings while it showed a peak on the 21st day in the control. PPO showed a peak on the 21st day in the IBA treated cuttings while it showed a peak on the 29th day in the control. SOD showed a peak on the 17th day in IBA treated cuttings, while it showed a peak on the 25th day in the control. Exogenous application of IBA enhanced the endogenous IAA and GA3 levels compared to CK, while it reduced the levels of ABA continuously at the time of rooting and then increased gradually. Inclusively, our study suggests that IBA 750 mg/L is efficient for the rooting of Magnolia biondii Pamp cuttings, as it enhanced the process of antioxidant enzyme activities, endogenous hormones levels and reduced the time of root formation which is evident from the anatomical observations.

Magnolia biondii Pamp é uma importante espécie de árvore ornamental muito cultivada e utilizada como porta-enxerto na propagação de diferentes variedades de Magnolia. Nos estudos atuais, hormônios anatômicos, fisiológicos e endógenos foram estudados para verificar o efeito do AIB na dose de 750 mg / L no enraizamento adventício e fornecer suporte teórico e técnico para a propagação de M. biondii Pamp por meio de estacas. Duas mil estacas foram preparadas e divididas em dois grupos, ou seja, tratadas com AIB e controle de água. Para a avaliação das atividades das enzimas antioxidantes e dos níveis de hormônios endógenos, as amostras foram coletadas no dia do plantio e a cada 5 dias, enquanto as demais etapas foram realizadas em laboratório de acordo com os protocolos e os devidos cuidados. Para as observações anatômicas, as amostras foram coletadas no 13º, 15º e 17º dias para estacas tratadas com AIB e no 21º, 23º e 25º dias para o controle. As amostras coletadas foram preservadas em solução FAA, e outras observações foram realizadas em laboratório. Observações anatômicas mostraram a necessidade de 13 dias para a diferenciação dos primórdios radiculares até o aparecimento de raízes adventícias jovens em estacas tratadas com AIB e de 21 dias para o desenvolvimento dos primórdios no controle. As atividades das enzimas antioxidantes envolvidas nas ROS foram significativamente maiores nas estacas tratadas com AIB em comparação com o controle. A POD apresentou pico no 13º dia antes da emergência das raízes nas estacas tratadas com AIB, enquanto no 21º dia apresentou pico no controle. A PPO teve pico no 21º dia nas estacas tratadas com AIB e no 29º dia no controle. A SOD apresentou pico no 17º dia nas estacas tratadas com AIB e no 25º dia no controle. A aplicação exógena de AIB aumentou os níveis endógenos de IAA e GA3 em relação ao controle, enquanto reduziu os níveis de ABA continuamente no momento do enraizamento e, em seguida, aumentou gradativamente. Inclusive, nosso estudo sugere que o AIB na dose de 750 mg / L é eficiente para o enraizamento de estacas de M. biondii Pamp, visto que potencializou o processo de atividades de enzimas antioxidantes e os níveis de hormônios endógenos, além de reduzir o tempo de formação de raízes, o que fica evidente nas observações anatômicas.

Examining the association of habitual e-cigarette use with inflammation and endothelial dysfunction in young adults: The VAPORS-Endothelial function study.

INTRODUCTION: Acute exposure to e-cigarette aerosol has been shown to have potentially deleterious effects on the cardiovascular system. However, the cardiovascular effects of habitual e-cigarette use have not been fully elucidated. Therefore, we aimed to assess the association of habitual e-cigarette use with endothelial dysfunction and inflammation - subclinical markers known to be associated with increased cardiovascular risk. METHODS: In this cross-sectional study, we analyzed data from 46 participants (23 exclusive e-cigarette users; 23 non-users) enrolled in the VAPORS-Endothelial function study. E-cigarette users had used e-cigarettes for ≥6 consecutive months. Non-users had used e-cigarettes <5 times and had a negative urine cotinine test (<30 ng/mL). Flow-mediated dilation (FMD) and reactive hyperemia index (RHI) were used to assess endothelial dysfunction, and we assayed high-sensitivity C-reactive protein, interleukin-6, fibrinogen, p-selectin, and myeloperoxidase as serum measures of inflammation. We used multivariable linear regression to assess the association of e-cigarette use with the markers of endothelial dysfunction and inflammation. RESULTS: Of the 46 participants with mean age of 24.3 ± 4.0 years, the majority were males (78%), non-Hispanic (89%), and White (59%). Among non-users, 6 had cotinine levels <10 ng/mL while 17 had levels 10-30 ng/mL. Conversely, among e-cigarette users, the majority (14 of 23) had cotinine ≥500 ng/mL. At baseline, the systolic blood pressure was higher among e-cigarette users than non-users (p=0.011). The mean FMD was slightly lower among e-cigarette users (6.32%) compared to non-users (6.53%). However, in the adjusted analysis, current e-cigarette users did not differ significantly from non-users in their mean FMD (Coefficient=2.05; 95% CI: -2.52-6.63) or RHI (Coefficient= -0.20; 95% CI: -0.88-0.49). Similarly, the levels of inflammatory markers were generally low and did not differ between e-cigarette users and non-users. CONCLUSIONS: Our findings suggest that e-cigarette use may not be significantly associated with endothelial dysfunction and systemic inflammation in relatively young and healthy individuals. Longer term studies with larger sample sizes are needed to validate these findings.

Feasibility and acceptability of bubble continuous positive airway pressure oxygen therapy for the treatment of childhood severe pneumonia and hypoxaemia in Bangladeshi children.

Background: Effective management of hypoxaemia is key to reducing pneumonia deaths in children. In an intensive care setting within a tertiary hospital in Bangladesh, bubble continuous positive airway pressure (bCPAP) oxygen therapy was beneficial in reducing deaths in this population. To inform a future trial, we investigated the feasibility of introducing bCPAP in this population in non-tertiary/district hospitals in Bangladesh. Methods: We conducted a qualitative assessment using a descriptive phenomenological approach to understand the structural and functional capacity of the non-tertiary hospitals (Institute of Child and Mother Health and Kushtia General Hospital) for the clinical use of bCPAP. We conducted interviews and focus group discussions (23 nurses, seven physicians, 14 parents). We retrospectively (12 months) and prospectively (three months) measured the prevalence of severe pneumonia and hypoxaemia in children attending the two study sites. For the feasibility phase, we enrolled 20 patients with severe pneumonia (age two to 24 months) to receive bCPAP, putting in place safeguards to identify risk. Results: Retrospectively, while 747 of 3012 (24.8%) children had a diagnosis of severe pneumonia, no pulse oxygen saturation information was available. Of 3008 children prospectively assessed with pulse oximetry when attending the two sites, 81 (3.7%) had severe pneumonia and hypoxaemia. The main structural challenges to implementation were the inadequate number of pulse oximeters, lack of power generator backup, high patient load with an inadequate number of hospital staff, and inadequate and non-functioning oxygen flow meters. Functional challenges were the rapid turnover of trained clinicians in the hospitals, limited post-admission routine care for in-patients by hospital clinicians due to their extreme workload (particularly after official hours). The study implemented a minimum of four hourly clinical reviews and provided oxygen concentrators (with backup oxygen cylinders), and automatic power generator backup. Twenty children with a mean age of 6.7 (standard deviation (SD) = 5.0)) months with severe pneumonia and hypoxaemia (median (md) SpO2 = 87% in room air, interquartile range (IQR) = 85-88)) with cough (100%) and severe respiratory difficulties (100%) received bCPAP oxygen therapy for a median of 16 hours (IQR = 6-16). There were no treatment failures or deaths. Conclusions: Implementation of low-cost bCPAP oxygen therapy is feasible in non-tertiary/district hospitals when additional training and resources are allocated.

Micro-dispenser-based optical packaging scheme for grating couplers.

Due to their sub-millimeter spatial resolution, ink-based additive manufacturing tools are typically considered less attractive than nanophotonics. Among these tools, precision micro-dispensers with sub-nanoliter volumetric control offer the finest spatial resolution: down to 50 µm. Within a sub-second, a flawless, surface-tension-driven spherical shape of the dielectric dot is formed as a self-assembled µlens. When combined with dispersive nanophotonic structures defined on a silicon-on-insulator substrate, we show that the dispensed dielectric µlenses [numerical aperture (NA) = 0.36] engineer the angular field distribution of vertically coupled nanostructures. The µlenses improve the angular tolerance for the input and reduces the angular spread of the output beam in the far field. The micro-dispenser is fast, scalable, and back-end-of-line compatible, allowing geometric-offset-caused efficiency reductions and center wavelength drift to be easily fixed. The design concept is experimentally verified by comparing several exemplary grating couplers with and without a µlens on top. A difference of less than 1 dB between incident angles of 7° and 14° is observed in the index-matched µlens, while the reference grating coupler shows around 5 dB contrast.

Pod-based e-cigarette use among US college-aged adults: A survey on the perception of health effects, sociodemographic correlates, and interplay with other tobacco products.

INTRODUCTION: E-cigarette use among youth and young adults remains of public health concern. Pod-based e-cigarettes, including JUUL, significantly changed the e-cigarette landscape in the US. Using an online survey, we explored the socio-behavioral correlates, predisposing factors, and addictive behaviors, among young adult pod-mod users within a University in Maryland, USA. METHODS: In total, 112 eligible college students aged 18-24 years, recruited from a University in Maryland, who reported using pod-mods were included in this study. Participants were categorized into current/non-current users based on past-30-day use. Descriptive statistics were used to analyze participants' responses. RESULTS: The mean age of the survey participants was 20.5 ± 1.2 years, 56.3% were female, 48.2% White, and 40.2% reported past-30-day (current) use of pod-mods. The mean age of first experimentation with pod-mods was 17.8 ± 1.4 years, while the mean age of regular use was 18.5 ± 1.4 years, with the majority (67.9%) citing social influence as the reason for initiation. Of the current users, 62.2% owned their own devices, and 82.2% predominantly used JUUL and menthol flavor (37.8%). A significant proportion of current users (73.3%) reported buying pods in person, 45.5% of whom were aged <21 years. Among all participants, 67% had had a past serious quit attempt. Among them, 89.3% neither used nicotine replacement therapy nor prescription medications. Finally, current use (adjusted odds ratio, AOR=4.52; 95% CI: 1.76-11.64), JUUL use (AOR=2.56; 95% CI: 1.08-6.03), and menthol flavor (AOR=6.52; 95% CI: 1.38-30.89) were associated with reduced nicotine autonomy, a measure of addiction. CONCLUSIONS: Our findings provide specific data to inform the development of public health interventions targeted at college youth, including the need for more robust cessation support for pod-mod users.

Analysis of serum calcium, sodium, potassium, zinc, and iron in patients with pre-eclampsia in Bangladesh: A case-control study.

Background and Aims: Pre-eclampsia is a particular type of pregnancy condition. Although the primary etiology of pre-eclampsia is unclear, it hypothesizes that the alteration of trace elements and macro-minerals may play a crucial function in the pathogenesis of Pre-eclampsia. Therefore, our research sought to ascertain the serum level of trace elements (zinc, iron) and macro-minerals (sodium, calcium, potassium) and their possible association with pre-eclampsia. Methods: The present study was conducted with 74 pre-eclampsia pregnant women (case) and 118 pregnant women having normal blood pressure (controls). Atomic Absorption Spectroscopy determined the serum level of trace components and electrolytes. Results: The researchers discovered notable differences in maternal age, gestational period, body mass index, systolic and diastolic blood pressure, hemoglobin, and creatinine level. Results of serum analysis revealed that calcium (52.06 ± 3.71 mg/L vs. 65.93 ± 2.57 mg/L, p < 0.05) and potassium (63.44 ± 5.33 mg/L vs. 102.54 ± 4.25 mg/L, p < 0.001) concentrations were substantially lower in the patient group than in control. Serum zinc (0.34 ± 0.02 mg/L vs. 0.52 ± 0.02 mg/L, p < 0.001) and iron (0.38 ± 0.03 mg/L vs. 0.46 ± 0.02 mg/L, p < 0.05) concentration were also considerably decreased in pre-eclampsia participants compared with a pregnant normotensive group. Pearson's correlation research results in the patient group revealed a connection between trace elements or macro minerals. In addition, the systolic blood pressure was positively correlated with sodium (r = 0.392, p < 0.01) and negatively correlated with potassium (r = -0.257, p < 0.05) in the control group. Conclusions: This study concludes that calcium, potassium, iron, and zinc levels were lower, whereas sodium levels were higher in Bangladeshi pre-eclampsia patients compared to controls. These findings with Pearson's correlation and the inter-element relationship between the patient and a control subject results can act as critical indication factors for patients with pre-eclampsia in Bangladesh and, as a result, may require a higher intake of calcium, potassium, iron, and zinc for effective therapeutic intervention and reduce the intake of sodium.

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor.

The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

The inverse association of state cannabis vaping prevalence with the e-cigarette or vaping product-use associated lung injury.

The e-cigarette or vaping product-use-associated lung injury (EVALI) epidemic was primarily associated with the use of e-cigarettes containing tetrahydrocannabinol (THC)- the principal psychoactive substance in cannabis, and vitamin-E-acetate- an additive sometimes used in informally sourced THC-containing e-liquids. EVALI case burden varied across states, but it is unclear whether this was associated with state-level cannabis vaping prevalence. We, therefore, used linear regression models to assess the cross-sectional association between state-level cannabis vaping prevalence (obtained from the 2019 behavioral Risk Factor Surveillance System) and EVALI case burden (obtained from the Centers for Disease Control and Prevention) adjusted for state cannabis policies. Cannabis vaping prevalence ranged from 1.14%(95%CI, 0.61%-2.12%) in Wyoming to 3.11%(95%CI, 2.16%-4.44%) in New Hampshire. EVALI cases per million population ranged from 1.90(0.38-3.42) in Oklahoma to 59.10(19.70-96.53) in North Dakota. There was no significant positive association but an inverse association between state cannabis vaping prevalence and EVALI case burden (Coefficient, -18.6; 95%CI, -37.5-0.4; p-value, 0.05). Thus, state-level cannabis vaping prevalence was not positively associated with EVALI prevalence, suggesting that there may not be a simple direct link between state cannabis vaping prevalence and EVALI cases, but rather the relationship is likely more nuanced and possibly reflective of access to informal sources of THC-containing e-cigarettes.

Multi-Scale Attention-Guided Non-Local Network for HDR Image Reconstruction.

High-dynamic-range (HDR) image reconstruction methods are designed to fuse multiple Low-dynamic-range (LDR) images captured with different exposure values into a single HDR image. Recent CNN-based methods mostly perform local attention- or alignment-based fusion of multiple LDR images to create HDR contents. Depending on a single attention mechanism or alignment causes failure in compensating ghosting artifacts, which can arise in the synthesized HDR images due to the motion of objects or camera movement across different LDR image inputs. In this study, we propose a multi-scale attention-guided non-local network called MSANLnet for efficient HDR image reconstruction. To mitigate the ghosting artifacts, the proposed MSANLnet performs implicit alignment of LDR image features with multi-scale spatial attention modules and then reconstructs pixel intensity values using long-range dependencies through non-local means-based fusion. These modules adaptively select useful information that is not damaged by an object's movement or unfavorable lighting conditions for image pixel fusion. Quantitative evaluations against several current state-of-the-art methods show that the proposed approach achieves higher performance than the existing methods. Moreover, comparative visual results show the effectiveness of the proposed method in restoring saturated information from original input images and mitigating ghosting artifacts caused by large movement of objects. Ablation studies show the effectiveness of the proposed method, architectural choices, and modules for efficient HDR reconstruction.

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection, helminthiasis, thrombosis and neuropharmacological disorders.

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds' phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 µg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2'-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.

Lipoprotein(a) and Subclinical Vascular and Valvular Calcification on Cardiac Computed Tomography: The Atherosclerosis Risk in Communities Study.

Background Lipoprotein(a) (Lp(a)) is a potent causal risk factor for cardiovascular events and mortality. However, its relationship with subclinical atherosclerosis, as defined by arterial calcification, remains unclear. This study uses the ARIC (Atherosclerosis Risk in Communities Study) to evaluate the relationship between Lp(a) in middle age and measures of vascular and valvular calcification in older age. Methods and Results Lp(a) was measured at ARIC visit 4 (1996-1998), and coronary artery calcium (CAC), together with extracoronary calcification (including aortic valve calcium, aortic valve ring calcium, mitral valve calcification, and thoracic aortic calcification), was measured at visit 7 (2018-2019). Lp(a) was defined as elevated if >50 mg/dL and CAC/extracoronary calcification were defined as elevated if >100. Logistic and linear regression models were used to evaluate the association between Lp(a) and CAC/extracoronary calcification, with further stratification by race. The mean age of participants at visit 4 was 59.2 (SD 4.3) years, with 62.2% women. In multivariable adjusted analyses, elevated Lp(a) was associated with higher odds of elevated aortic valve calcium (adjusted odds ratio [aOR], 1.82; 95% CI, 1.34-2.47), CAC (aOR, 1.40; 95% CI, 1.08-1.81), aortic valve ring calcium (aOR, 1.36; 95% CI, 1.07-1.73), mitral valve calcification (aOR, 1.37; 95% CI, 1.06-1.78), and thoracic aortic calcification (aOR, 1.36; 95% CI, 1.05-1.77). Similar results were obtained when Lp(a) and CAC/extracoronary calcification were examined on continuous logarithmic scales. There was no significant difference in the association between Lp(a) and each measure of calcification by race or sex. Conclusions Elevated Lp(a) at middle age is significantly associated with vascular and valvular calcification in older age, represented by elevated CAC, aortic valve calcium, aortic valve ring calcium, mitral valve calcification, thoracic aortic calcification. Our findings encourage assessing Lp(a) levels in individuals with increased cardiovascular disease risk, with subsequent comprehensive vascular and valvular assessment where elevated.

Coronary Artery Calcium for Risk Stratification of Sudden Cardiac Death: The Coronary Artery Calcium Consortium.

BACKGROUND: Coronary artery calcium (CAC) is a marker of plaque burden. Whether CAC improves risk stratification for incident sudden cardiac death (SCD) beyond atherosclerotic cardiovascular disease (ASCVD) risk factors is unknown. OBJECTIVES: SCD is a common initial manifestation of coronary heart disease (CHD); however, SCD risk prediction remains elusive. METHODS: The authors studied 66,636 primary prevention patients from the CAC Consortium. Multivariable competing risks regression and C-statistics were used to assess the association between CAC and SCD, adjusting for demographics and traditional risk factors. RESULTS: The mean age was 54.4 years, 33% were women, 11% were of non-White ethnicity, and 55% had CAC >0. A total of 211 SCD events (0.3%) were observed during a median follow-up of 10.6 years, 91% occurring among those with baseline CAC >0. Compared with CAC = 0, there was a stepwise higher risk (P trend < 0.001) in SCD for CAC 100 to 399 (subdistribution hazard ratio [SHR]: 2.8; 95% CI: 1.6-5.0), CAC 400 to 999 (SHR: 4.0; 95% CI: 2.2-7.3), and CAC >1,000 (SHR: 4.9; 95% CI: 2.6-9.9). CAC provided incremental improvements in the C-statistic for the prediction of SCD among individuals with a 10-year risk <7.5% (ΔC-statistic = +0.046; P = 0.02) and 7.5% to 20% (ΔC-statistic = +0.069; P = 0.003), which were larger when compared with persons with a 10-year risk >20% (ΔC-statistic = +0.01; P = 0.54). CONCLUSIONS: Higher CAC burden strongly associates with incident SCD beyond traditional risk factors, particularly among primary prevention patients with low-intermediate risk. SCD risk stratification can be useful in the early stages of CHD through the measurement of CAC, identifying patients most likely to benefit from further downstream testing.

Assessment of heavy metals in cyprinid fishes: Rivers of district Khuzdar Balochistan Pakistan.

The present study was conducted to measured heavy metals in cyprinid fishes in rivers of District Khuzdar Balochistan, Pakistan. In the present study, 25 fish samples were collected that belonged to 8 order of 13 families, The Cyprinidae family had the largest number of eight fish species. Present study is focused on Heavy metals in cyprinid fishes. Heavy metals accumulation like Zinc, Manganese, Copper, and Nickel was evaluated in water and various organs of fishes. Atomic Absorption Spectroscopy was used for the identification of these heavy metals in fish species and water bodies. The average concentration (mg/L) of Zn 0.26-0.41, Mn 0.030- 0.073, Cu 0.017-0.080 and NI 0.14-0.79 were observed in water. The Concentration (mg/L), of Zn Conc 0.383-.028 Mn Conc .073- .030 Cu Conc 080-.017 NI Conc .79-.14. The concentration of heavy metals was found both similar and varied simultaneously across the whole research area. Zinc concentration was reported highest, whereas Copper was at the lowest concentration in all fish species .The concentration of heavy metals, in all the fish species under this study, was above the threshold of WHO limits.

Developing a Scale Measuring Patient Expectations and Service Quality of Hospitals in India during COVID-19.

This paper develops a scale that measures the perceived service quality of hospitals during a pandemic. To develop the scale, data from 206 respondents from India, was subjected to exploratory and confirmatory factor analysis. The newly developed scale was named PAND-SERVQUAL, which includes factors namely, assistance, facility & layout, trust, empathy, promptness, and knowledge. The resulting scale is likely to be useful for researchers exploring service quality research and health care quality as well. Findings will facilitate understanding patient's expectations regarding the service quality of hospitals during a pandemic.Supplemental data for this article is available online at https://doi.org/10.1080/00185868.2021.1939827 .

Aspirin for cardiovascular disease prevention among adults in the United States: Trends, prevalence, and participant characteristics associated with use.

OBJECTIVE: : Aspirin has been widely utilized over several decades for atherosclerotic cardiovascular disease (ASCVD) prevention among adults in the United States. We examined trends in aspirin use among adults aged ≥40 years from 1998 to 2019 and assessed factors associated with its use for primary and secondary ASCVD prevention. METHODS: : Using 1998-2019 Behavioral Risk Factor Surveillance System data, we obtained weighted prevalence of aspirin use among adults aged ≥40 years for each year and examined trends in use over this period. Using multivariable logistic regression and utilizing data from 54,388 respondents aged ≥40 years in the 2019 data, we assessed factors associated with aspirin use for secondary prevention and for primary prevention stratified by the number of traditional ASCVD risk factors reported (hypertension, diabetes mellitus, high cholesterol, overweight/obesity, and cigarette smoking). RESULTS: : Aspirin use prevalence increased from 29.0%(95%CI, 27.9%-30.2%) in 1998 to 37.5%(36.9%-38.0%) in 2009. However, use has slightly declined over the last decade: 35.6%(34.6%-36.6%) in 2011 to 33.5%(32.5%-34.6%) in 2019. In 2019, among respondents without cardiovascular disease (CVD), 27.5%(26.4%-28.6%) reported primary prevention aspirin use while 69.7%(67.0%-72.2%) of respondents with CVD reported secondary prevention aspirin use. Of concern, 45.6%(43.5%-47.7%) of adults aged ≥70 years without CVD reported primary prevention aspirin use. Additionally, among individuals without any self-reported traditional ASCVD risk factor, males (adjusted odds ratio(aOR):1.60, 95%CI:1.12-2.27), persons aged ≥70 years (aOR:3.22, 95%CI:2.27-4.55), and individuals with healthcare coverage (aOR:2.28, 95%CI:1.17-4.44) had higher odds of primary prevention aspirin use compared to females, persons aged 40-69 years, and individuals without healthcare coverage, respectively. Females were less likely than males to report secondary prevention aspirin use (aOR:0.64, 95%CI:0.50-0.82). CONCLUSION: : Aspirin use has slightly declined over the last decade. A significant proportion of adults aged ≥70 years reported primary prevention aspirin use in 2019. Since current guidelines do not recommend primary prevention aspirin use among adults aged ≥70 years, such use should be discouraged.