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Black carbon (BC) and brown carbon (BrC) both have a versatile nature, and they have an apparent role in climate variability and changes. As anthropogenic activity is surging, BC and BrC are also reportedly increasing. So, the monitoring of BC and BrC and observations of land use land cover change (LULCC) at a regional level are necessary for the changes in various interconnected meteorological phenomena. The current study investigates BC, BrC, CO2, BC from fossil fuels (BCff), BC from biomass burning (BCbb), and LULCC and their relationship to the corresponding meteorological conditions over Gangtok in the Sikkim Himalayan region. The concentration of BC (BrC) was found to be highest during March 2022 (April 2021) at 43.5 µg m−3 (32.0 µg m−3)​​​​​​​. Surface pressure exhibits a significant positive correlation with BC, BCff, BCbb, and BrC. Higher surface pressure results in a calmer and more stable boundary layer, which effectively retains deposited contaminants. Conversely, the wind appears to facilitate the dispersion of pollutants, showing a strong negative correlation. The fact that all pollutants and precipitation have been shown to behave similarly points to moist scavenging of the pollutants. Despite the dense cloud cover, it is clear that the area is not receiving convective precipitation, implying that orographic precipitation is occurring over the region. Most of Sikkim receives convective rain from May to September, indicating that the region has significant convective activity contributed from the Bay of Bengal during the monsoon season. Furthermore, monsoon months have the lowest concentrations of BC, BCbb, BCff, and BrC, suggesting the potential of convective rain (as rainout scavenging) to remove most of the pollutants.

Retinoyl fluoride (2) prepared from retinoic acid (1) by reaction with diethylaminosulfurtrifluoride is a stable crystalline compound not easily hydrolyzed by water. By reacting retinoyl fluoride with water-soluble amines in the presence of sodium bicarbonate, retinamide (4), N-retinoyl glycine (6), N-retinoyl DL-phenylalanine (7), alpha-N-retinoyl-L-lysine (11), N-retinoyl 4-aminophenol (4-hydroxyphenylretinamide) (8), and N-retinoyl-2-amino-2-deoxy-D-glucose (2-deoxy-D-glucose-2-retinamide) (9) have been prepared in good yields and characterized by UV absorption, 1H NMR, IR spectra, mass spectrometry, and elemental analysis.

All-trans retinoyl fluoride reacts with the 3,6-lactone of glucuronic acid in slightly alkaline solution to give the lactone of retinoyl beta-glucuronide, along with other retinoyl glucurono-lactones, in about 60% yield. Hydrolysis of the lactone with very dilute alkali gives the free acid, retinoyl beta-glucuronide, in about 80% yield. Pure all-trans retinoyl beta-glucuronide (overall yield: 20-25%) was obtained free from other isomeric and anomeric forms by reverse-phase high pressure liquid chromatography. Retinoyl beta-glucuronide was characterized by UV-visible, infrared, and 1H-NMR spectra, by elementary analysis, by mass spectra, and by its susceptibility to hydrolysis by bacterial beta-glucuronidase.

Oxidation of retinol at the C-4 position is a major metabolic route to biologically inactive excretory products. Thus, the replacement of hydrogen with fluorine at C-4 might well modify the rate of metabolism, biological activity, and pharmacological activity of vitamin A, 4,4-Difluororetinyl acetate and related analogs were consequently synthesized by the following procedure. Retinoic acid (1), upon methylation to methyl retinoate (2), was oxidized with manganese dioxide to methyl 4-oxoretinoate (3). Methyl 4,4-difluororetinoate (4), prepared from methyl 4-oxoretinoate (3) by reaction with diethylaminosulfurtrifluoride (DAST), was further converted to 4,4-difluororetinoic acid (5), 4,4-difluororetinol (6), 4,4-difluororetinyl acetate (7), and 4,4-difluororetinyl palmitate (8). 4,4-Difluororetinyl acetate was also prepared directly from 4-oxoretinyl acetate (9) by reaction with DAST. The purified, often crystalline, analogs were characterized by UV absorption, mass spectrometry, and 1H-NMR and 19F-NMR spectra.

Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer, with increasing global incidence and have poor prognosis. Tumour-infiltrating lymphocytes (TILs) are recognized as a key prognostic indicator and play a vital role in OSCC grading. However, current methods for TILs quantification are based on subjective visual assessments, leading to inter-observer variability and inconsistent diagnostic reproducibility. Only a few studies have been conducted in automating TILs quantification for OSCC, existing methods use score-based systems that focus only on tissue-level spatial analysis, overlooking essential cellular-level information and do not provide TILs infiltration subcategories required for determining OSCC grading. To address these limitations, we propose OralTILs-ViT, a novel joint representation learning framework that integrates cellular and tissue-level information. Our model employs two parallel encoders: one extracts cellular features from cellular density maps, while the other processes tissue features from H&E-stained tissue images. This dual-encoder approach enables OralTILs-ViT to capture complex tissue-cellular interactions, classifying TILs infiltration categories consistent with Broders' grading system-"Moderate to Marked", "Slight" and "None to Very Less." This approach reflects pathology practices and increases TILs classification accuracy. To generate cellular density maps, we introduce TILSeg-MobileViT, a multiclass segmentation model trained using a weakly supervised method, minimizing the need for manual annotation of cellular masks and overcoming the limitations of previous TILs assessment techniques. An extensive evaluation of our methodology demonstrates that OralTILs-ViT with the configuration (Adam, α = 0.001) outperforms existing approaches, achieving 96.37% accuracy, 96.34% precision, 96.37% recall, and a 96.35% F1 score. Furthermore, TOPSIS analysis confirms that our method ranks first across all TILs infiltration categories. In summary, our proposed methodology outperforms single modality-representation learning approaches for accurate and automated TILs classification., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Several studies on noncommunicable diseases (NCDs) have been carried out worldwide, the basis of most of which is the identification of risk factors-modifiable (or behavioral) and metabolic. Majority of the NCDs are due to sociodemographic factors, lifestyle, and behavior, which can be prevented to a great extent. Thus, it is a health challenge and a necessity to identify such factors of NCDs., Objectives: The objective is to make a thorough systematic and comparative analysis of diverse machine learning (ML) classifiers and identify the best-performing model to study social determinants of NCDs., Materials and Methods: We used data from the Longitudinal Ageing Study in India, and predicted the prevalence of NCDs based on a set of sociodemographic, lifestyle, and behavioral risk factors by conducting a comparative analysis among 25 different algorithms., Results: Evaluating the performance metrics, the random forest model was found to be the most-suited method with 87.9% accuracy and hence chosen as the final model for the analysis. The model's performance was optimized by a hyper-parameter tuning process using grid-search with a 5-fold cross-validation strategy and results suggested that it was able to make accurate predictions on new instances., Conclusion: The epidemic of chronic illness cannot be completely addressed without comprehending the social determinants. With advancements in medical and health-care industry, ML has been applied to analyze diseases based on clinical parameters. This work is an attempt by the authors to explore and encourage the use of ML in the field of social epidemiology., (Copyright © 2024 Copyright: © 2024 Indian Journal of Public Health.)

Increasing contamination in potable water supplies necessitates the development of sensing methods that provide the speed and selectivity necessary for safety. One promising method relies on recognition and detection at the liquid-liquid interface of dynamic complex emulsions. These all-liquid materials transduce changes in interfacial tensions into optical signals via the coupling of their chemical, physical, and optical properties. Thus, to introduce selectivity, it is necessary to modify the liquid-liquid interface with an interfacially stable and selective recognition unit. To this end, we report the synthesis and characterization of amphiphilic block copolymers modified with metal chelators to selectively measure the concentrations of dissolved metal ions. We find that significant reduction in interfacial tensions arises upon quantitative addition of metal ions with high affinity toward functionalized chelators. Furthermore, measurements from UV-vis spectroscopy reveal that complexation of the block copolymers with metal ions leads to an increase in surface excess and surfactant effectiveness. We also demonstrate selective detection of iron(III) cations (Fe 3+ ) on the μM levels even through interference from other mono-, di-, or trivalent cations in complex matrices of synthetic groundwater. Our results provide a unique platform that couples selective recognition and modulation of interfacial behaviors and demonstrates a step forward in the development of the multiplexed sensing device needed to deconvolute the complicated array of contaminants that comprise real-world environmental samples.

Oral Squamous Cell Carcinoma (OSCC) is the most prevalent type of oral cancer across the globe. Histopathology examination is the gold standard for OSCC examination, where stained histopathology slides help in studying and analyzing the cell structures under a microscope to determine the stages and grading of OSCC. One of the staining methods popularly known as H&E staining is used to produce differential coloration, highlight key tissue features, and improve contrast, which makes cell analysis easier. However, the stained H&E histopathology images exhibit inter and intra-variation due to staining techniques, incubation times, and staining reagents. These variations negatively impact computer-aided diagnosis (CAD) and Machine learning algorithm's accuracy and development. A pre-processing procedure called stain normalization must be employed to reduce stain variance's negative impacts. Numerous state-of-the-art stain normalization methods are introduced. However, a robust multi-domain stain normalization approach is still required because, in a real-world situation, the OSCC histopathology images will include more than two color variations involving several domains. In this paper, a multi-domain stain translation method is proposed. The proposed method is an attention gated generator based on a Conditional Generative Adversarial Network (cGAN) with a novel objective function to enforce color distribution and the perpetual resemblance between the source and target domains. Instead of using WSI scanner images like previous techniques, the proposed method is experimented on OSCC histopathology images obtained by several conventional microscopes coupled with cameras. The proposed method receives the L* channel from the L*a*b* color space in inference mode and generates the G(a*b*) channel, which are color-adapted. The proposed technique uses mappings learned during training phases to translate the source domain to the target domain; mapping are learned using the whole color distribution of the target domain instead of one reference image. The suggested technique outperforms the four state-of-the-art methods in multi-domain OSCC histopathological translation, the claim is supported by results obtained after assessment in both quantitative and qualitative ways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Emissive complex droplets with reconfigurable morphology and dynamic optical properties offer exciting opportunities as chemical sensors due to their stimuli-responsive characteristics. In this work, we demonstrated a real-time optical sensing platform that combines poly(dimethylsiloxane) (PDMS) microfluidics and complex droplets as sensing materials. We utilized a mechanism, called directional emission, to transduce changes in interfacial tension into optical signals. We discuss the fabrication and integration of PDMS microfluidics with complex emulsions to facilitate continuous measurement of fluorescent emission and, ultimately, the interfacial tensions. Furthermore, by varying the interfacial functionalization and fluorescent dye with characteristic wavelength, we generate multiple formulations of droplets and obtain differential responses to stimuli that alter interfacial tensions ( i.e. , composition of surfactants, pH). Our results illustrate a proof-of-concept multiplexed and continuous sensing platform with potential applications in miniaturized, on-site environmental monitoring and biosensing.

Competing Interests: The authors have declared that no competing interests exist.

A cross-sectional study was conducted with 144 small-scale poultry farmers across 42 Bangladeshi villages to explore risk factors associated with avian influenza H5 and H9 seropositivity on backyard chicken farms. Using mixed-effects logistic regression with village as random effect, we identified crow abundance in garbage dumping places and presence of migratory wild birds within villages to be associated with higher odds of H5 and H9 seropositivity. At farm-level, garbage around poultry houses was also associated with higher odds of H5 and H9 seropositivity. In addition, specific trading practices (such as, purchase of chickens from live bird markets (LBM) and neighboring farms to raise them on their own farms, frequency of visits to LBM, purchase of poultry at LBM for consumption) and contact of backyard chickens with other animals (such as, feeding of different poultry species together, using pond water as drinking source for poultry, access of feral and wild animals to poultry houses) were associated with higher odds of H5 or H9 seropositivity. Resource-constrained small-scale poultry farmers should be able to address risk factors identified in this study without requiring large investments into poultry management, thereby reducing the likelihood of avian influenza virus transmission and ultimately occurrence of avian influenza outbreaks., (© 2022. The Author(s).)

Accurate and timely monitoring is imperative to the resilience of forests for economic growth and climate regulation. In the UK, forest management depends on citizen science to perform tedious and time-consuming data collection tasks. In this study, an unmanned aerial vehicle (UAV) equipped with a light sensor and positioning capabilities is deployed to perform aerial surveying and to observe a series of forest health indicators (FHIs) which are inaccessible from the ground. However, many FHIs such as burrows and deadwood can only be observed from under the tree canopy. Hence, we take the initiative of employing a quadruped robot with an integrated camera as well as an external sensing platform (ESP) equipped with light and infrared cameras, computing, communication and power modules to observe these FHIs from the ground. The forest-monitoring time can be extended by reducing computation and conserving energy. Therefore, we analysed different versions of the YOLO object-detection algorithm in terms of accuracy, deployment and usability by the EXP to accomplish an extensive low-latency detection. In addition, we constructed a series of new datasets to train the YOLOv5x and YOLOv5s for recognising FHIs. Our results reveal that YOLOv5s is lightweight and easy to train for FHI detection while performing close to real-time, cost-effective and autonomous forest monitoring.

Objectives: We aimed at the identification of the association of comorbidities with the COVID-19 severity and hospitalization., Methods: It is a retrospective cross-sectional study to investigate the variation in age, sex, dwelling, comorbidities, and medication with the COVID-19 severity and hospitalization by enrolling 1025 recovered individuals while comparing their time of recovery with or without comorbidities., Results: COVID-19 patients mostly suffered from fever. The predominant underlying medical conditions in them were hypertension (HTN) followed by diabetes mellitus (DM). Patients with cardiovascular disease (CVD) (54.3%) and hepatic disorders (HD) (43.6%) experienced higher severity. The risk of symptomatic cases was higher in aged (odds ratio, OR = 1.04, 95% CI = 1.02-1.06) and comorbid (OR = 1.87, 95% CI = 1.34-2.60) patients. T-test confirmed the differences between the comorbid and non-comorbid patients' recovery duration. The presence of multiple comorbidities increased the time of recovery (15-27 days) and hospitalization (20-40%). Increased symptomatic cases were found for patients having DM+HTN whereas CVD+Asthma patients were found with higher percentage of severity. Besides, DM+CKD (chronic kidney disease) was associated with higher hospitalization rate. Higher odds of severity were found for DM+CVD (OR = 4.42, 95% CI = 1.81-10.78) patients. Hospitalization risk was also increased for them (OR = 5.14, 95% CI = 2.02-13.07). Moreover, if they had HTN along with DM+CVD, they were found with even higher odds (OR = 6.82, 95% CI = 2.37-19.58) for hospitalization., Conclusion: Our study indicates that people who are aged, females, living in urban area and have comorbid conditions are at a higher risk for developing COVID-19 severity. Clinicians and health management authorities should prioritize these high-risk groups to reduce mortality attributed to the disease., (Copyright: © International Journal of Health Sciences.)

Sepsis is a major cause of mortality among hospitalized patients worldwide. Rapid diagnosis is critical as early treatments have been demonstrated to improve survival. Despite the importance of early detection, current technologies and clinical methods are often insufficient due to their lack of the necessary speed, selectivity, or sensitivity. The development of rapid sensing platforms that target sepsis-related biomarkers could significantly improve the outcomes of patients. This Mini-Review focuses on the recent advances in rapid diagnosis of soluble biomarkers in blood with the emphasis on different configurations of point-of-care (POC) instruments. Specifically, it first describes the commonly targeted biomarkers and the mechanisms by which they are detected. Then, it highlights the recently developed sensors that aim to reduce the total time of diagnosis without sacrificing selectivity and limit of detection. These sensors are categorized based on their distinct sensing and transduction mechanisms. Finally, it concludes with a brief outlook over future developments of multiplexed sensors., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Hypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout β-cardiac myosin, including in the motor domain, are associated with HCM. A β-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near-normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal β-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM., Competing Interests: AS, BB, JA, HS, EF, YG, DW, EO No competing interests declared, (© 2021, Snoberger et al.)

The COVID-19 pandemic is damaging economies across the world, including financial markets and institutions in all possible dimensions. For banks in particular, the pandemic generates multifaceted crises, mostly through increases in default rates. This is likely to be worse in developing economies with poor financial market architecture. This paper utilizes Bangladesh as a case study of an emerging economy and examines the possible impacts of the pandemic on the country's banking sector. Bangladesh's banking sector already has a high level of non-performing loans (NPLs) and the pandemic is likely to worsen the situation. Using a state-designed stress testing model, the paper estimates the impacts of the COVID-19 pandemic on three particular dimensions-firm value, capital adequacy, and interest income-under different NPL shock scenarios. Findings suggest that all banks are likely to see a fall in risk-weighted asset values, capital adequacy ratios, and interest income at the individual bank and sectoral levels. However, estimates show that larger banks are relatively more vulnerable. The decline in all three dimensions will increase disproportionately if NPL shocks become larger. Findings further show that a 10% NPL shock could force capital adequacy of all banks to go below the minimum BASEL-III requirement, while a shock of 13% or more could turn it to zero or negative at the sectoral level. Findings call for immediate and innovative policy measures to prevent a large-scale and contagious banking crisis in Bangladesh. The paper offers lessons for other developing and emerging economies similar to Bangladesh., Competing Interests: Conflict of interestThey have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© Springer Nature Switzerland AG 2020.)

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin's working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Our purpose of the study was to assess the knowledge and any gaps of early educators regarding hearing impairment, and to attempt to delineate the attitudes and perceptions of the educators regarding the outcomes of their students with hearing disabilities and their views on inclusive teaching, using a questionnaire based study. Only 71 respondents completed the questionnaire. The present questionnaire based study was conducted on 100 primary school teachers, undertaken on randomly selected schools from Gangtok, East Sikkim. Their knowledge and attitudes regarding hearing impairment in children in the areas of cause, diagnosis, investigations, treatment, prevention, special care and inclusion into mainstream schools were assessed. Our study showed that the knowledge of causes, investigation and treatment of hearing impairement [HI] among respondents/teachers was poor. The data was shown statistically significant with P  < 0.001. However, responses testing behavior and attitudes towards HI was encouraging. 52.9% respondents disagreed that HI was a social handicap and 88.2% felt that teachers need special training before a HI child is admitted to the school. The results were statistically significant with P  < 0.001. The biggest obstacle found for inclusive education of HI children was a lack of proper training to teachers (40.8%) and attitude of other students (29.5%)., Competing Interests: The authors declare that there is no conflict of interests.This article does not contain any studies with animals performed by any of the authors. All procedures performed in this study involving human participant was in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from the participants included in the study.

Muscle contraction, cytokinesis, cellular movement, and intracellular transport depend on regulated actin-myosin interaction. Most actin filaments bind one or more isoform of tropomyosin, a coiled-coil protein that stabilizes the filaments and regulates interactions with other actin-binding proteins, including myosin. Isoform-specific allosteric regulation of muscle myosin II by actin-tropomyosin is well-established while that of processive myosins, such as myosin V, which transport organelles and macromolecules in the cell periphery, is less certain. Is the regulation by tropomyosin a universal mechanism, the consequence of the conserved periodic structures of tropomyosin, or is it the result of specialized interactions between particular isoforms of myosin and tropomyosin? Here, we show that striated muscle tropomyosin, Tpm1.1, inhibits fast skeletal muscle myosin II but not myosin Va. The non-muscle tropomyosin, Tpm3.1, in contrast, activates both myosins. To decipher the molecular basis of these opposing regulatory effects, we introduced mutations at conserved surface residues within the six periodic repeats (periods) of Tpm3.1, in positions homologous or analogous to those important for regulation of skeletal muscle myosin by Tpm1.1. We identified conserved residues in the internal periods of both tropomyosin isoforms that are important for the function of myosin Va and striated myosin II. Conserved residues in the internal and C-terminal periods that correspond to Tpm3.1-specific exons inhibit myosin Va but not myosin II function. These results suggest that tropomyosins may directly impact myosin function through both general and isoform-specific mechanisms that identify actin tracks for the recruitment and function of particular myosins., (© 2018 Wiley Periodicals, Inc.)

Timely diagnosis of tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is only achieved for ~58% cases. An improved, accurate, time- and cost-effective method for bacteriological confirmation of MTB is necessary. We evaluated Mycotube, a new variant of Lowenstein-Jensen (LJ) culture medium, by comparing it with Mycobacterium Growth Indicator Tube (MGIT) 960 (gold standard), local LJ, and bioMérieux LJ-T in terms of isolation rate and time-to-growth. Pulmonary and extra-pulmonary samples from treatment-naïve suspects (n = 207) were decontaminated by the N-acetyl-L-cysteine-sodium hydroxide method and used to inoculate the four media. Subjective and objective parameters were used for evaluation. Mycotube yielded 140 positive results, compared to 162, 69, and 141 from MGIT, local LJ, and LJ-T, respectively. Of these, 139 (67%) were true-positive results and 1 (0.5%) was false-positive. The mean time-to-growth detection was 17.4 days for Mycotube, compared to 14.5, 28.1, and 16.5 days for MGIT, local LJ, and LJ-T, respectively. The mean time-to-growth for local LJ significantly differed from that for MGIT, but not those for LJ-T and Mycotube. No contamination was observed. Mycotube had a sensitivity of 85.8% and a specificity of 97.8% as compared to MGIT. Mycotube offers good results, comparable with those observed for conventional LJ. It requires only basic laboratory infrastructure. The overall cost of the test should be nearly three times lower than that of MGIT. Mycotube helps with TB diagnosis and generates pure isolates for drug susceptibility testing.

Elephant endotheliotropic herpesviruses (EEHVs) are the cause of acute hemorrhagic disease in endangered Asian and African elephants. In the present study, we report the incidence of EEHV infection and associated mortality in the captive elephant of Assam, India. Our result showed the gross morphology and histopathological changes of EEHV infection in the elephant. Moreover, the phylogenetic analysis of the polymerase, helicase, and GPCR genes from the infected tissue samples suggested the presence of EEHV1A virus., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tropomyosin is the archetypal-coiled coil, yet studies of its structure and function have proven it to be a dynamic regulator of actin filament function in muscle and non-muscle cells. Here we review aspects of its structure that deviate from canonical leucine zipper coiled coils that allow tropomyosin to bind to actin, regulate myosin, and interact directly and indirectly with actin-binding proteins. Four genes encode tropomyosins in vertebrates, with additional diversity that results from alternate promoters and alternatively spliced exons. At the same time that periodic motifs for binding actin and regulating myosin are conserved, isoform-specific domains allow for specific interaction with myosins and actin filament regulatory proteins, including troponin. Tropomyosin can be viewed as a universal regulator of the actin cytoskeleton that specifies actin filaments for cellular and intracellular functions.

Tropomyosin (Tpm) is a two-chained α-helical coiled-coil protein that binds to filamentous actin (F-actin), and regulates its interactions with myosin by occupying three average positions on F-actin (blocked, closed, and open). Mutations in the Tpm are linked to heart diseases including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). To elucidate the molecular mechanisms of Tpm mutations (including DCM mutation E54K, HCM mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K, and a designed synthetic mutation D137L) in terms of their effects on Tpm flexibility and its interactions with F-actin, we conducted extensive molecular dynamics simulations for the wild-type and mutant Tpm in complex with F-actin (total simulation time 160 ns per mutant). The mutants exhibited distinct changes (i.e., increase or decrease) in the overall and local flexibility of the Tpm coiled-coil, with each mutation causing both local and long-range modifications of the Tpm flexibility. In addition, our binding calculations revealed weakened Tpm-F-actin interactions (except for L185R, D137L and A63V) involving five periods of Tpm, which correlate with elevated fluctuation of Tpm relative to the blocked position on F-actin that may lead to easier activation and increased Ca 2+ -sensitivity. We also simulated the αβ/βα-Tpm heterodimer in comparison with the αα-Tpm homodimer, which revealed greater flexibility and weaker actin binding in the heterodimer. Our findings are consistent with a complex mechanism underlying how different Tpm mutations perturb the Tpm function in distinct ways (e.g., by affecting specific sites of Tpm), which bear no simple links to the disease phenotypes (e.g., HCM vs. DCM).

A new class of multidentate dipyrrolylmethane based ditopic tecton, 1,9-bis(3,5-dimethylpyrazolylmethyl)dipyrrolylmethane, containing diethyl (L1) or cyclohexylidene (L2) substituents at the meso carbon atom were readily synthesized in 28-45% yields in two different ways starting from dipyrrolylmethanes. A one dimensional coordination polymer structure ([(L2)Ag][BF4])n was obtained when L2 was treated with AgBF4, whereas the analogous reaction between L1 and AgBF4 afforded the dicationic binuclear metallacycle complex [(L1)2Ag2][BF4]2. In addition, yet another coordination polymeric structure [(L1)CuI]n was obtained from the reaction between L1 and CuI. The analogous reaction of L1 with [Pd(PhCN)2Cl2] afforded the binuclear palladium complex [(L1)2Pd2Cl4] having a double-stranded helicate structure. The observed structural differences are attributed to the effects of the substituents present at the meso carbon atom of the ligand, in addition to the nature of the metal centre, coordination number and the preferred geometry.

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9-7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, α-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

Laparoscopic cystogastrostomy is an established procedure for the drainage of pancreatic pseudocysts. Cysts are mainly present in the lesser sac (retro-gastric), which is completely amenable to cystogastrostomy. We discuss the problems faced and simple solutions to the problems in managing a huge pancreatic pseudocyst of 22 cm×18 cm in a young boy 18 y of age.

Toxin-antitoxin (TA) systems are found on both bacterial plasmids and chromosomes, but in most cases their functional role is unclear. Gene knockouts often yield limited insights into functions of individual TA systems because of their redundancy. The well-characterized F-plasmid-based CcdAB TA system is important for F-plasmid maintenance. We have isolated several point mutants of the toxin CcdB that fail to bind to its cellular target, DNA gyrase, but retain binding to the antitoxin, CcdA. Expression of such mutants is shown to result in release of the WT toxin from a functional preexisting TA complex as well as derepression of the TA operon. One such inactive, active-site mutant of CcdB was used to demonstrate the contribution of CcdB to antibiotic persistence. Transient activation of WT CcdB either by coexpression of the mutant or by antibiotic/heat stress was shown to enhance the generation of drug-tolerant persisters in a process dependent on Lon protease and RecA. An F-plasmid containing a ccd locus can, therefore, function as a transmissible persistence factor.

Background: Pectus excavatum (PE) is the most common chest wall deformity in adolescent life. Nuss procedure is a well-established technique for the repair of PE. The indication for correction is mainly medical aesthetic. Advantages of Nuss over conventional methods include reduced length of hospital stay, smaller incisions, and absence of need for osteochondrectomies. Here, we describe our experience with this procedure., Materials and Methods: This was a retrospective study of patients who underwent Nuss procedure by a single surgeon between 2006 and 2010 in a regional center. Indications for surgery included the following: Progressive deformity and psychological stress. All patients underwent chest X-ray and pulmonary function testing. A standard Nuss procedure was performed using a single bar. Patients' satisfaction was assessed by a questionnaire and follow-up clinic letters. Satisfaction with body image was scored on a scale of 1-10., Results: Eleven patients with PE underwent correction by Nuss procedure. Mean age of the patient was 19 years (range: 15-30). The average hospital stay was 7 days (range: 4-23 days). There was no mortality and no episodes of wound infection. In the immediate post-operative period, three patients (12.5%) were noted to have poor pain control. The post-operative course was uneventful in all cases except one patient who developed lung collapse, pleural effusion, and bar dislocation. Hundred percent of patients were satisfied with the scar. Seven patients scored 7 out of 10 on satisfaction with body image and two patients scored 6 or less. None of the patients complained of chronic pain., Conclusion: Nuss procedure is an effective method for the correction of PE. Most patients were satisfied with the outcome and none experienced chronic pain.

We investigate the suitability of the two existing risk stratification systems available for predicting mortality in a cohort of patients undergoing lung resection under a single surgeon. Data from the 290 consecutive patients who underwent pulmonary resection between January 2008 and January 2011 were extracted from a prospective clinical data base. In-hospital mortality risk scores are calculated for every patient by using Thoracoscore and ESOS.01 and were compared with actual in-hospital mortality. The receiver operating characteristic (ROC) curve was used to establish how well the systems rank for predicting patient mortality. Actual in-hospital mortality was 3.1% (n = 9). Thoracoscore and ESOS values (mean ± SEM) were 4.93 ± 0.32 and 4.08 ± 0.41, respectively. The area under the ROC curve values for ESOS and Thoracoscore were 0.8 and 0.6, respectively. ESOS was reasonably accurate at predicting the overall mortality (sensitivity 88% and specificity 67%), whereas Thoracoscore was a weaker predictor of mortality (sensitivity 67% and specificity 53%). The ESOS score had better predictive values in our patient population and might be easier to calculate. Because of their low specificity, the use of these scores should be limited to the assessment of outcomes of surgical cohorts, but they are not designed to predict risks for individual patients.

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors., (© 2011 American Chemical Society)

Tropomyosin (Tm) is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3-4 sites mutated to Ala within the first half or the second half of periods 2-6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (> 4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤ 2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.

Background: Refractory post cardiopulmonary bypass (CPB) bleeding continues to cause concern for cardiac surgeons and intensivists. Massive postoperative hemorrhage following CPB is multifactorial and not fully understood, and it is also associated with increased mortality and morbidity. Activated recombinant factor VII (rFVIIa) has emerged as possible salvage medication in refractory post cardiac surgical bleeding. This observational study sought to identify the pattern of use of rFVIIa in cardiac surgery, its effectiveness, and risk., Methods: This study involved a retrospective case review of medical records of ten patients undergoing a variety of cardiac surgery procedures and who developed life-threatening bleeding during surgery or after surgery despite conventional medical therapy, including transfusion of blood and blood products, and received rFVIIa at a regional center between August 2007 and April 2009., Results: All ten patients received two consecutive doses of rFVIIa (average dose 65 μg/kg) at a 2-hour interval. Eight patients were re-explored due to massive postoperative bleeding or cardiac tamponade before receiving rFVIIa. Surgical sources of bleeding were not identified in any cases. A second re-exploration was carried out in two cases. Two patients (20%) died in ITU from problems not related to bleeding and thromboembolism. Blood loss was significantly reduced after administration of rFVIIa. Blood loss 6 hours prior to treatment was 1758.5 ± 163.9 mL and blood loss in the 6-hour period post treatment was 405.6 ± 50.5 mL (P < 0.05). Blood and blood products used in the 6-hour period before and after administration of rFVIIa were 19.6 ± 1.5U and 4.4 ± 0.6U, respectively (P < 0.05). No adverse reactions or thrombotic complications related to rFVIIa were noted., Conclusion: In our limited study, use of rFVIIa in refractory post surgical bleeding was significantly reduced blood loss and use of blood and blood products. We concluded that rFVIIa can be used satisfactorily and safely as a rescue therapy in the management of post cardiac surgical bleeding.

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.

The folding enantioselectivity for D-Ala versus L-Ala at one glycine site in the Trp-cage is 16 kJ mol(-1); judicious introductions of alanines of the correct chirality raises the melting temperature of this 20-residue fold to 83 degrees C.

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

Alginate, a calcium-sensitive polymer, could carry out simultaneous purification and refolding of 8 M urea/100 mM dithiothreitol (DTT) denatured and thermally denatured alpha-amylase present in a commercial preparation. Activity recoveries of 80 and 70% in the former and the latter cases, respectively, were obtained. The fluorescence spectra showed refolding, and PAGE showed the absence of any aggregates in the refolded preparation. As another example, Eudragit S-100, a pH-sensitive poly(methyl methacrylate), was used to refold CcdB (controller of cell division or death B) protein. Initial experiments with wild-type (WT) CcdB showed that Eudragit bound and precipitated (upon lowering the pH to 4.0) CcdB quantitatively from the latter's aqueous solution. The bioconjugate showed DNA gyrase inhibition activity of CcdB and could be recycled. The inclusion bodies of CcdB mutant CcdB-17P were solubilized in 8 M urea/100 mM dithiothreitol. This preparation could be refolded by precipitation with Eudragit. The fluorescence and CD spectra showed that protein refolding has occurred.