Targeting STING oligomerization with small-molecule inhibitors.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Dual Inhibitors of Main Protease (MPro) and Cathepsin L as Potent Antivirals against SARS-CoV2.

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Proximity-Dependent Labeling of Cysteines.

Mapping protein-protein interactions is crucial for understanding various signaling pathways in living cells, and developing new techniques for this purpose has attracted significant interest. Classic methods (e.g., the yeast two-hybrid) have been supplanted by more sophisticated chemical approaches that label proximal proteins (e.g., BioID, APEX). Herein we describe a proximity-based approach that uniquely labels cysteines. Our approach exploits the nicotinamide N-methyltransferase (NNMT)-catalyzed methylation of an alkyne-substituted 4-chloropyridine (SS6). Upon methylation of the pyridinium nitrogen, this latent electrophile diffuses out of the active site and labels proximal proteins on short time scales (≤5 min). We validated this approach by identifying known (and novel) interacting partners of protein arginine deiminase 2 (PAD2) and pyruvate dehydrogenase kinase 1 (PDK1). To our knowledge, this technology uniquely exploits a suicide substrate to label proximal cysteines in live cells.

Are all charge-transfer parameters created equally? A study of functional dependence and excited-state charge-transfer quantification across two dye families.

Small molecule organic dyes have many potential uses in medicine, textiles, forensics, and light-harvesting technology. Being able to computationally predict the spectroscopic properties of these dyes could greatly expedite screening efforts, saving time and materials. Time-dependent density functional theory (TD-DFT) has been shown to be a good tool for this in many instances, but characterizing electronic excitations with charge-transfer (CT) character has historically been challenging and can be highly sensitive to the chosen exchange-correlation functional. Here we present a combined experimental and computational study of the excited-state electronic structure of twenty organic dyes obtained from the Max Weaver Dye Library at NCSU. Results of UV-vis spectra calculations on these dyes with six different exchange-correlation functionals, BP86, B3LYP, PBE0, M06, BH and HLYP, and CAM-B3LYP, were compared against their measured UV-vis spectra. It was found that hybrid functionals with modest amounts (20-30%) of included Hartree-Fock exchange are the most effective at matching the experimentally determined λmax. The interplay between the observed error, the functional chosen, and the degree of CT was analyzed by quantifying the CT character of λmax using four orbital and density-based metrics, Λ, Δr, SC and DCT, as well as the change in the dipole moment, Δµ. The results showed that the relationship between CT character and the functional dependence of error is not straightforward, with the observed behavior being dependent both on how CT was quantified and the functional groups present in the molecules themselves. It is concluded that this may be a result of the examined excitations having intermediate CT character. Ultimately it was found that the nature of the molecular "family" influenced how a given functional behaved as a function of CT character, with only two of the examined CT quantification methods, Δr and DCT, showing consistent behavior between the different molecular families. This suggests that further work needs to be done to ensure that currently used CT quantification methods show the same general trends across large sets of multiple dye families.

Interprofessional Development of a Livestream Simulation Activity to Enhance an Undergraduate Nursing Research Course.

The article describes the development, implementation, and evaluation of an interactive simulation activity to enhance student engagement and comprehension of evidence-based practice principles. An interprofessional team of nurse educators, simulation experts, information technology specialists, and nursing informatics graduate students collaborated on the simulation design. The results of this project support the need to develop innovative learning strategies to facilitate nursing students' understanding of the relevance of evidence-based practice research to improve patient outcomes.

Feasibility of Employing mHealth in Delivering Preventive Nutrition Interventions Targeting the First 1000 Days of Life: Experiences from a Community-Based Cluster Randomised Trial in Rural Bangladesh.

BACKGROUND/OBJECTIVES: An Android platform-based customised app and web-linked system was developed to aid in implementing selected nutrition interventions by community health workers (CHWs) in a community-based cluster randomised trial (c-RCT) in rural Bangladesh. METHODS: Here, we describe the architecture of the intervention delivery system, and explore feasibility of employing mHealth as CHWs' job aid, employing a mixed-method study design covering 17 visits per mother-child dyad. We analysed CHWs' real-time visit information from monitoring and documentation data, and CHWs' qualitative interviews to explore the advantages and barriers of using mHealth as a job aid. RESULTS: Intervention coverage was high across the arms (>90%), except around the narrow perinatal period (51%) due to mothers' cultural practice of moving to their parents' homes and/or hospitals for childbirth. CHWs mentioned technical and functional advantages of the job aid including device portability, easy navigability of content, pictorial demonstration that improved communication, easy information entry, and automated daily scheduling of tasks. Technical challenges included charging tablets, especially in power cut-prone areas, deteriorated battery capacity over continuous device usage, unstable internet network in unsupportive weather conditions, and device safety. Nevertheless, onsite supervision and monitoring by expert supervisors remained important to ensure intervention quality. CONCLUSIONS: With appropriate training and supervision, CHWs utilised the tablet-based app proficiently, attaining high coverage of long-term visits. mHealth was thus useful for designing, planning, scheduling, and delivering nutrition interventions through CHWs, and for monitoring and supervision by supervisors. Therefore, this application and job aid can be adopted or replicated into the currently developing national health systems platform for improving coverage and quality of preventive maternal and child nutrition services. In addition, continuous supportive supervision by skilled supervisors must be accompanied to ensure CHWs' task quality. Finally, future studies should rigorously assess undesirable health and environmental effects of mHealth before and after mainstreaming, effective interventions addressing device-induced health hazards should be designed and scaled up, and effective e-waste management must be ensured.

Performance of chest X-ray scoring in predicting disease severity and outcomes of patients hospitalised with COVID-19 in Bangladesh.

Introduction: Evaluation of potential outcomes of COVID-19-affected pneumonia patients using computed tomography scans may not be conceivable in low-resource settings. Thus, we aimed to evaluate the performance of chest X-ray scoring in predicting the disease severity and outcomes of adults hospitalised with COVID-19. Methods: This was a retrospective chart analysis consuming data from COVID-19-positive adults who had chest X-ray availability and were admitted to a temporary COVID unit, in Bangladesh from 23rd April 2020 to 15th November 2021. At least one clinical intensivist and one radiologist combinedly reviewed each admission chest X-ray for the different lung findings. Chest X-ray scoring varied from 0 to 8, depending on the area of lung involvement with 0 indicating no involvement and 8 indicating ⩾75% involvement of both lungs. The receiver operating characteristic curve was used to determine the optimum chest X-ray cut-off score for predicting the fatal outcomes. Result: A total of 218 (82.9%) out of 263 COVID-19-affected adults were included in the study. The receiver operating characteristic curve demonstrated the optimum cut-off as ⩾3 and ⩾5 for disease severity and death, respectively. In multivariate logistic regression analysis, a chest X-ray score of ⩾3 was found to be independently associated with disease severity (aOR: 8.70; 95% CI: 3.82, 19.58, p < 0.001) and a score of ⩾5 with death (aOR: 16.53; 95% CI: 4.74, 57.60, p < 0.001) after adjusting age, sex, antibiotic usage before admission, history of fever, cough, diabetes mellitus, hypertension, total leukocytes count and C-reactive protein. Conclusion: Using chest X-ray scoring derived cut-off at admission might help to identify the COVID-19-affected adults who are at risk of severe disease and mortality. This may help to initiate early and aggressive management of such patients, thereby reducing their fatal outcomes.

ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na+/K+ transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and biochemical assessments. Proteomic analysis of immunoprecipitated ATP1A3+ brain-derived EVs shows higher enrichment of synaptic markers and cargo proteins relevant to Alzheimer's disease (AD) compared to NCAM1+ or LICAM+ EVs. Single particle analysis shows the elevated amyloid-ß positivity in ATP1A3+ EVs from AD plasma, providing better diagnostic prediction of AD over other plasma biomarkers. Thus, ATP1A3 is a reliable target to isolate NDEV from biofluids for diagnostic research.

Trends and correlates of low HIV knowledge among ever-married women of reproductive age: Evidence from cross-sectional Bangladesh Demographic and Health Survey 1996-2014.

BACKGROUND: The human immunodeficiency virus (HIV) burden has frequently been changing over time due to epidemiological and demographic transitions. To safeguard people, particularly women of reproductive age, who can be exposed to transmitting this burden to the next generation, knowledge regarding this life-threatening virus needs to be increased. This research intends to identify the trends and associated correlates of "low" HIV knowledge among ever-married women of reproductive age in Bangladesh from 1996 to 2014. METHODS: We analyzed data derived from six surveys of Bangladesh Demographic and Health Surveys conducted in 1996, 1999, 2004, 2007, 2011, and 2014. Analyses were primarily restricted to ever-married women aged 15-49 years who had ever heard of HIV. The correlates of "low" HIV knowledge were investigated using multiple binary logistic regression models. RESULTS: The study found that the proportion of women with "low" HIV knowledge decreased from 72% in 1996 to 58% in 2014. In adjusted models, age at first marriage, level of education, wealth quintile, and place of residence (except in the survey year 2011) were found to be potential correlates of "low" HIV knowledge in all survey years. In the pooled analysis, we found lower odds of "low" HIV knowledge in the survey years 1999 (Adjusted Odds Ratio: 0.67; 95% CI: 0.57, 0.78), 2004 (AOR: 0.60; 95% CI: 0.52, 0.70), 2007 (AOR: 0.51; 95% CI: 0.44, 0.60), 2011 (AOR: 0.36; 95% CI: 0.32, 0.42) and 2014 (AOR: 0.47; 95% CI: 0.41, 0.54) compared to the survey year 1996. CONCLUSION: The proportion of "low" HIV knowledge has declined over time, although the proportion of women with "low" HIV knowledge still remains high. The prevention of early marriage, the inclusion of HIV-related topics in the curricula, reduction of disparities between urban-rural and the poorest-richest groups may help to improve the level of HIV knowledge among ever-married Bangladeshi women.

Prevalence and predictors of magnesium imbalance among critically ill diarrheal children and their outcome in a developing country.

Despite having essential roles in maintaining human body physiology, magnesium has gained little attention. We sought to evaluate the prevalence and predictors of magnesium imbalance in diarrheal children admitted to an intensive care unit. This retrospective data analysis was conducted among children admitted between January 2019 and December 2019. Eligible children were categorized by serum magnesium levels that were extracted from the hospital database. Among 557 participants, 29 (5.2%) had hypomagnesemia, 344 (61.8%) had normomagnesemia and 184 (33.0%) had hypermagnesemia. By multivariable multinomial logistic regression, we have identified older children (adjusted multinomial odds ratio, mOR 1.01, 95% CI: 1.004-1.018, p = 0.002) as a predictor of hypomagnesemia. Conversely, younger children (adjusted mOR 0.99, 95% CI: 0.982-0.998, p = 0.02), shorter duration of fever (adjusted mOR 0.92, 95% CI: 0.857-0.996, p = 0.04), convulsion (adjusted mOR 1.55, 95% CI: 1.005-2.380, p = 0.047), dehydration (adjusted mOR 3.27, 95% CI: 2.100-5.087, p<0.001), pneumonia (adjusted mOR 2.65, 95% CI: 1.660-4.240, p<0.001) and acute kidney injury (adjusted mOR 2.70, 95% CI: 1.735-4.200, p<0.001) as the independent predictors of hypermagnesemia. The mortality was higher among children with hypermagnesemia (adjusted mOR 2.31, 95% CI: 1.26-4.25, p = 0.007). Prompt identification and management of the magnesium imbalance among critically ill diarrheal children might have survival benefits, especially in resource-limited settings.

Socioeconomic Inequalities in Women's Undernutrition: Evidence from Nationally Representative Cross-Sectional Bangladesh Demographic and Health Survey 2017-2018.

The objective of this study was to explore the socioeconomic inequalities in undernutrition among ever-married women of reproductive age. We used nationally representative cross-sectional data from the Bangladesh Demographic and Health Survey, 2017−2018. Undernutrition was defined as a body mass index (BMI) of <18.5 kg/m2. The concentration index (C) was used to measure the socioeconomic inequality in the prevalence of women's undernutrition. A multiple binary logistic regression model was carried out to find out the factors associated with women's undernutrition. The prevalence of undernutrition among women of 15−49 years was 12%. Among them, 8.5% of women were from urban and 12.7% of women were from rural areas. The prevalence of undernutrition was highest (21.9%) among women who belonged to the adolescent age group (15−19 years). The C showed that undernutrition was more prevalent among the socioeconomically worst-off (poorest) group in Bangladesh (C = −0.26). An adjusted multiple logistic regression model indicated that women less than 19 years of age had higher odds (adjusted odds ratio, AOR: 2.81; 95% confidence interval, CI: 2.23, 3.55) of being undernourished. Women from the poorest wealth quintile (AOR: 3.93, 95% CI: 3.21, 4.81) had higher odds of being undernourished. On the other hand, women who had completed secondary or higher education (AOR: 0.55; 95% CI: 0.49, 0.61), married women who were living with their husbands (AOR: 0.72, 95% CI: 0.61, 0.86), and women exposed to mass media (AOR: 0.87, 95% CI: 0.79, 0.97) were less likely to be undernourished. Intervention strategies should be developed targeting the poorest to combat undernutrition in women of reproductive age in Bangladesh.

Using stable isotope tracers to monitor membrane dynamics in C. elegans.

Membranes within an animal are composed of phospholipids, cholesterol, and proteins that together form a dynamic barrier. The types of lipids that are found within a membrane bilayer impact its biophysical properties including its fluidity, permeability, and susceptibility to damage. While membrane composition is very stable in healthy adults, aberrant membrane structure is seen in a wide and varied array of diseases as well as during natural aging. Despite the wide-reaching impacts of membrane composition, there is relatively little known about how membrane landscape is established and maintained over time. In vivo biochemical modeling of membrane lipids is needed to understand how these molecules interact in their natural configurations. Here, we have described analytical methods that increase the capacity to map the dynamics of individual membrane phospholipids using different types of mass spectrometry. Specifically, we describe novel stable isotope (13C and 15N) strategies to quantify the turnover of dozens of fatty acid tails and intact phospholipids simultaneously.

Microfluidic preparation, shrinkage, and surface modification of monodispersed alginate microbeads for 3D cell culture.

Functionalized alginate microbeads (MB) have been widely used for three-dimensional (3D) culture of cells and creating biomimetic tissue models. However, conventional methods for preparing these MB suffer from poor polydispersity, due to coalescence of droplets during the gelation process and post-aggregation. It remains an immense challenge to prepare alginate MB with narrow size distribution and uniform shape, especially when their diameters are similar to the size of cells. In this work, we developed a simple method to produce monodispersed, cell-size alginate MB through microfluidic emulsification, followed by a controlled shrinkage process and gelation in mineral oil with low concentration of calcium ion (Ca2+). During the gelation process caused by the diffusion of Ca2+ from the oil to water phase, a large amount of satellite droplets with sub-micrometer sizes was formed at the water/oil interface. As a result, each original droplet was transformed to one shrunken-MB with much smaller size and numerous submicron-size satellites. To explore the feasibility of the shrunken-MB for culturing with cells, we have successfully modified a variety of polymer nanofilms on MB surfaces using a layer-by-layer assembly approach. Finally, the nanofilm-modified MB was applied to a 3D culture of GFP-expressing fibroblast cells and demonstrated good biocompatibility.

Stimulation-dependent remodeling of the corticospinal tract requires reactivation of growth-promoting developmental signaling pathways.

The corticospinal tract (CST) can become damaged after spinal cord injury or stroke, resulting in weakness or paralysis. Repair of the damaged CST is limited because mature CST axons fail to regenerate, which is partly because the intrinsic axon growth capacity is downregulated in maturity. Whereas CST axons sprout after injury, this is insufficient to recover lost functions. Chronic motor cortex (MCX) electrical stimulation is a neuromodulatory strategy to promote CST axon sprouting, leading to functional recovery after CST lesion. Here we examine the molecular mechanisms of stimulation-dependent CST axonal sprouting and synapse formation. MCX stimulation rapidly upregulates mTOR and Jak/Stat signaling in the corticospinal system. Chronic stimulation, which leads to CST sprouting and increased CST presynaptic sites, further enhances mTOR and Jak/Stat activity. Importantly, chronic stimulation shifts the equilibrium of the mTOR repressor PTEN to the inactive phosphorylated form suggesting a molecular transition to an axon growth state. We blocked each signaling pathway selectively to determine potential differential contributions to axonal outgrowth and synapse formation. mTOR blockade prevented stimulation-dependent axon sprouting. Surprisingly, Jak/Stat blockade did not abrogate sprouting, but instead prevented the increase in CST presynaptic sites produced by chronic MCX stimulation. Chronic stimulation increased the number of spinal neurons expressing the neural activity marker cFos. Jak/Stat blockade prevented the increase in cFos-expressing neurons after chronic stimulation, confirming an important role for Jak/Stat signaling in activity-dependent CST synapse formation. MCX stimulation is a neuromodulatory repair strategy that reactivates distinct developmentally-regulated signaling pathways for axonal outgrowth and synapse formation.

Direct analysis of textile dyes from trace fibers by automated microfluidics extraction system coupled with Q-TOF mass spectrometer for forensic applications.

Textile fiber is a common form of transferable trace evidence at the crime scene. Different techniques such as microscopy or spectroscopy are currently being used for trace fiber analysis. Dye characterization in trace fiber adds an important molecular specificity during the analysis. In this study, we performed a direct trace fiber analysis method via dye characterization by a novel automated microfluidics device (MFD) dye extraction system coupled with a quadrupole-time-of-flight (Q-TOF) mass spectrometer (MS). The MFD system used an in-house made automated procedure which requires only 10µL of organic solvent for the extraction. The total extraction and identification time by the system is under 12min. A variety of sulfonated azo and anthraquinone dyes were analyzed from ∼1mm length nylon fiber samples. This methodology successfully characterized multiple dyes (≥3 dyes) from a single fiber thread. Additionally, it was possible to do dye characterization from single fibers with a diameter of ∼10µm. The MFD-MS system was used for elemental composition and isotopic distribution analysis where MFD-MS/MS was used for structural characterization of dyes on fibers.

Weaver's historic accessible collection of synthetic dyes: a cheminformatics analysis.

We present the Max Weaver Dye Library, a collection of ∼98 000 vials of custom-made and largely sparingly water-soluble dyes. Two years ago, the Eastman Chemical Company donated the library to North Carolina State University. This unique collection of chemicals, housed in the College of Textiles, also includes tens of thousands of fabric samples dyed using some of the library's compounds. Although the collection lies at the core of hundreds of patented inventions, the overwhelming majority of this chemical treasure trove has never been published or shared outside of a small group of scientists. Thus, the goal of this donation was to make this chemical collection, and associated data, available to interested parties in the research community. To date, we have digitized a subset of 2700 dyes which allowed us to start the constitutional and structural analysis of the collection using cheminformatics approaches. Herein, we open the discussion regarding the research opportunities offered by this unique library.

Genetic Analysis of Avian Influenza Viruses: Cocirculation of Avian Influenza Viruses with Allele A and B Nonstructural Gene in Northern Pintail (Anas acuta) Ducks Wintering in Japan.

The pandemic influenza virus strains of 1918 (H1N1), 1957 (H2N2), 1968 (H3N2), and 2009 (H1N1) have genes related to avian influenza viruses (AIVs). The nonstructural (NS) gene of AIVs plays a significant role in host-viral interaction. However, little is known about the degree of diversity of this gene in Northern pintail (Anas acuta) ducks wintering in Japan. This study describes characteristics of pintail-originated H1N1, H1N2, H1N3, H5N2, H5N3, H5N9, and H7N7 viruses. Most of the viruses were revealed to be avian strains and not related to pandemic and seasonal flu strains. Nevertheless, the NP genes of 62.5% (5/8) viruses were found closely related to a A/swine/Korea/C12/08, indicating exchange of genetic material and ongoing mammalian-linked evolution of AIVs. Besides, all the viruses, except Aomori/422/07 H1N1, contain PSIQSR∗GLF motif usually found in avian, porcine, and human H1 strains. The Aomori/422/07 H1N1 has a PSVQSR∗GLF motif identical to a North American strain. This findings linked to an important intercontinental, Asian-American biogeographical interface. Phylogenetically all the viruses were clustered in Eurasian lineage. Cocirculation of allele A and B (NS gene) viruses was evident in the study implying the existence of a wide reservoir of influenza A viruses in pintail wintering in Japan.

Evaluation of human intestinal epithelial differentiated cells (Caco-2) for replication, plaque formation and isolation of avian influenza viruses.

Although various cultured cells are used for propagating influenza A viruses, the types of cells which can support replication of and plaque production by low pathogenic avian influenza (LPAI) viruses without supplementary trypsin are limited. In this study, the infectivity and growth kinetics of as well as plaque production by LPAI viruses in Caco-2 cells were investigated. The suitability of this cell line for virus isolation was examined and compared with virus isolation in embryonated chicken eggs. Generation of Caco-2 mediated viral variants, if any, was assessed phenotypically and genotypically. It was found that Caco-2 cells can readily support continued replication of LPAI viruses without supplementary trypsin. Viruses replicate to high titer compared to embryonated chicken eggs, and more efficiently than in MDCK cells, without trypsin. Also, LPAI viruses produced plaques in Caco-2 cells. However, these cells were found to be less sensitive than embryonated chicken eggs for virus isolation. Notably, no phenotypic and genotypic changes of the viruses were observed during viral passages (at least up to 10th passage) in Caco-2 cells. These findings indicate that Caco-2 cells may provide an appropriate substrate for studying and cultivating AIVs.