Human Coronavirus Infection Reorganizes Spatial Genomic Architecture in Permissive Lung Cells.

Chromatin conformation capture followed by next-generation sequencing in combination with large-scale polymer simulations (4DHiC) produces detailed information on genomic loci interactions, allowing for the interrogation of 3D spatial genomic structures. Here, Hi-C data was acquired from the infection of fetal lung fibroblast (MRC5) cells with α-coronavirus 229E (CoV229E). Experimental Hi-C contact maps were used to determine viral-induced changes in genomic architecture over a 48-hour time period following viral infection, revealing substantial alterations in contacts within chromosomes and in contacts between different chromosomes. To gain further structural insight and quantify the underlying changes, we applied the 4DHiC polymer simulation method to reconstruct the 3D genomic structures and dynamics corresponding to the Hi-C maps. The models successfully reproduced experimental Hi-C data, including the changes in contacts induced by viral infection. Our 3D spatial simulations uncovered widespread chromatin restructuring, including increased chromosome compactness and A-B compartment mixing arising from infection. Our model also suggests increased spatial accessibility to regions containing interferon-stimulated genes upon infection with CoV229E, followed by chromatin restructuring at later time points, potentially inducing the migration of chromatin into more compact regions. This is consistent with previously observed suppression of gene expression. Our spatial genomics study provides a mechanistic structural basis for changes in chromosome architecture induced by coronavirus infection in lung cells.

Activation of automethylated PRC2 by dimerization on chromatin.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit EZH2 stimulates its activity by an unknown mechanism. Here, we show that PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a novel mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification coupled dimerization in the regulation of chromatin modifying complexes.

Utility of VCS Parameters as a Cost-effective and Early Marker of Sepsis: A Hospital-based Study.

Introduction: Sepsis is a life-threatening condition. Nowadays, hospitals rely on laboratory parameters like CRP and procalcitonin to detect sepsis. There is a need to evaluate and validate more accurate and early predictors of sepsis in critically ill patients. We analyzed volume, conductivity, and scatter (VCS) parameters of leukocytes in sepsis patients and compared them with the control group with no illness. Materials and methods: It was a prospective case-control study. A total of 80 patients were studied with 40 sepsis cases and 40 controls. Peripheral smear examination was done in all the cases. Other parameters, such as WBC count, neutrophil%, absolute neutrophil count (ANC), High-sensitive C-reactive protein (hs-CRP), procalcitonin, and blood cultures were analyzed. We took the data of the patients from medical records and correlated it with other tests. Complete blood picture reports were generated by the Beckman Coulter LH series (LH 750 and 780). VCS parameters for neutrophils, lymphocytes and monocytes were compared between both groups. The results were analyzed using SPSS software (16.0 version). Results: The age group was 20-85 years with male predominance. The mean neutrophil volume (MNV) and mean monocyte volume (MMV) were higher in the sepsis group when compared with the control group. The mean neutrophil conductivity and scatter was lower in the sepsis group and comparatively higher in the control group, but it was insignificant. The mean neutrophil volume values were higher in the sepsis group even with low total leukocyte count (TLC) when the patient had sepsis. There is no change in the mean lymphocyte VCS parameter and mean monocyte conductivity, scatter in both the groups. Conclusion: The mean neutrophil volume and MMV are found to be good sensitive markers in the prediction of sepsis. Even when TLC is on the lower side in patients with sepsis, these parameters predict sepsis accurately. This helps clinicians to assess sepsis in patients at an early stage and has an important practical implication. How to cite this article: Goyal H, Singhal A, Joseph M. Utility of VCS Parameters as a Cost-effective and Early Marker of Sepsis: A Hospital-based Study. Indian J Crit Care Med 2023;27(9):647-650.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

Natural History of Asymptomatic Walled-off Necrosis in Patients With Acute Pancreatitis.

Background and objectives Studies on the natural history of asymptomatic walled-off necrosis (WON) in acute pancreatitis (AP) are scarce. We conducted a prospective observational study to look for the incidence of infection in WON. Material and methods In this study, we included 30 consecutive AP patients with asymptomatic WON. Their baseline clinical, laboratory, and radiological parameters were recorded and followed up for three months. Mann Whitney U test and unpaired t-tests were used for quantitative data and chi-square and Fisher's exact tests were used for qualitative data analysis. A p-value <0.05 was considered significant. Receiver operating characteristic curve (ROC) analysis was done to identify the appropriate cutoffs for the significant variables. Results Of the 30 patients enrolled, 25 (83.3%) were males. Alcohol was the most common etiology. Eight patients (26.6%) developed an infection on follow-up. All were managed by drainage either percutaneously (n=4, 50%) or endoscopically (n=3, 37.5%). One patient required both. No patient required surgery and there was no mortality. Median baseline C-reactive protein (CRP) was higher in infection group 76 (IQR=34.8) mg/L vs asymptomatic group, 9.5 mg/dl (IQR=13.6), p<0.001. IL-6 and tumor necrosis factor (TNF)-alpha was also higher in the infection group. The size of the largest collection (157.50±33.59 mm vs 81.95±26.22 mm, P<0.001) and CT severity index (CTSI) (9.50±0.93 vs 7.82±1.37, p<0.01) were also higher in infection group as compared to the asymptomatic group. ROC curve analysis of baseline CRP (cutoff 49.5mg/dl), size of WON (cutoff 127mm) and CTSI (cutoff of 9) showed AUROC (area under ROC) of 1, 0.97, and 0.81 respectively for the future development of infection in WON. Conclusion Around one-fourth of asymptomatic WON patients developed an infection during three-months follow-up. Most patients with infected WON can be managed conservatively.

A Core-Shell Approach for Systematically Coarsening Nanoparticle-Membrane Interactions: Application to Silver Nanoparticles.

The continuous release of engineered nanomaterial (ENM) into the environment may bring about health concerns following human exposure. One important source of ENMs are silver nanoparticles (NPs) that are extensively used as anti-bacterial additives. The introduction of ENMs into the human body can occur via ingestion, skin uptake or the respiratory system. Therefore, evaluating how NPs translocate over bio-membranes is essential in assessing their primary toxicity. Unfortunately, data regarding membrane-NP interaction is still scarce, as is theoretical and in silico insight into what governs adhesion and translocation for the most relevant NPs and membranes. Coarse-grained (CG) molecular descriptions have the potential to alleviate this situation, but are hampered by the absence of a direct link to NP materials and membrane adhesion mechanisms. Here, we interrogate the relationship between the most common NP representation at the CG level and the adhesion characteristics of a model lung membrane. We find that this representation for silver NPs is non-transferable, meaning that a proper CG representation for one size is not suited for other sizes. We also identify two basic types of primary adhesion-(partial) NPs wrapping by the membrane and NP insertion into the membrane-that closely relate to the overall NP hydrophobicity and significantly differ in terms of lipid coatings. The proven non-transferability of the standard CG representation with size forms an inspiration for introducing a core-shell model even for bare NPs that are uniform in composition. Using existing all-atom molecular dynamics (MD) data as a reference, we show that this extension does allow us to reproduce size-dependent NP adhesion properties and lipid responses to NP binding at the CG level. The subsequent CGMD evaluation for 10 nm Ag NPs provides new insight into membrane binding for relevant NP sizes and into the role of water in trapping NPs into defected mixed monolayer-bilayer states. This development will be instrumental for simulating NP-membrane adhesion towards more experimentally relevant length and time scales for particular NP materials.

Use of Uncertainty of Measurement for Traceability of Test Results and Setting up of own Quality Goal for Methods having Lower Stability- A Tertiary Care Hospital study.

Uncertainty of measurement (UM) provides a quantitative estimate for traceability of test results. The Nordtest guide was applied for calculating UM of 26 analytes. For this, internal and external quality control data from July 2019 to April 2020 was used. UM of test results were compared to %TEa values of CLIA '2019, RiliBÄK, and Ricos. It was observed that UM for all analytes were below %TEa values of RiliBÄK. UM value of Albumin, Calcium and Sodium could not meet CLIA '2019 and Ricos guidelines. For results of Albumin, Calcium and Sodium to be traceable, more frequent quality control protocols resulted in decrease in bias. Quality goals were set for these three parameters. This helped in reduction of quality control cycles and optimum utilization of resources.

Predicted Adsorption Affinity for Enteric Microbial Metabolites to Metal and Carbon Nanomaterials.

Ingested nanomaterials are exposed to many metabolites that are produced, modified, or regulated by members of the enteric microbiota. The adsorption of these metabolites potentially affects the identity, fate, and biodistribution of nanomaterials passing the gastrointestinal tract. Here, we explore these interactions using in silico methods, focusing on a concise overview of 170 unique enteric microbial metabolites which we compiled from the literature. First, we construct quantitative structure-activity relationship (QSAR) models to predict their adsorption affinity to 13 metal nanomaterials, 5 carbon nanotubes, and 1 fullerene. The models could be applied to predict log k values for 60 metabolites and were particularly applicable to 'phenolic, benzoyl and phenyl derivatives', 'tryptophan precursors and metabolites', 'short-chain fatty acids', and 'choline metabolites'. The correlations of these predictions to biological surface adsorption index descriptors indicated that hydrophobicity-driven interactions contribute most to the overall adsorption affinity, while hydrogen-bond interactions and polarity/polarizability-driven interactions differentiate the affinity to metal and carbon nanomaterials. Next, we use molecular dynamics (MD) simulations to obtain direct molecular information for a selection of vitamins that could not be assessed quantitatively using QSAR models. This showed how large and flexible metabolites can gain stability on the nanomaterial surface via conformational changes. Additionally, unconstrained MD simulations provided excellent support for the main interaction types identified by QSAR analysis. Combined, these results enable assessing the adsorption affinity for many enteric microbial metabolites quantitatively and support the qualitative assessment of an even larger set of complex and biologically relevant microbial metabolites to carbon and metal nanomaterials.

Elucidation of SARS-Cov-2 Budding Mechanisms through Molecular Dynamics Simulations of M and E Protein Complexes.

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides.

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from K F ≈ 108 to 104 M-1), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting 'K' peptides and homodimer-forming 'E' peptides. Furthermore, a new and highly fusogenic CC pair (E3/P1K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2E/P2K and P3E/P3K. This E3/P1k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.

The role of size and nature in nanoparticle binding to a model lung membrane: an atomistic study.

Understanding the uptake of nanoparticles (NPs) by different types of cellular membranes plays a pivotal role in the design of NPs for medical applications and in avoiding adverse effects that result in nanotoxicity. Yet, the role of key design parameters, such as the bare NP material, NP size and surface reactivity, and the nature of NP coatings, in membrane remodelling and uptake mechanisms is still very poorly understood, particularly towards the lower range of NP dimensions that are beyond the experimental imaging resolution. The same can be said about the role of a particular membrane composition. Here, we systematically employ biased and unbiased molecular dynamics simulations to calculate the binding energy for three bare materials (Ag/SiO2/TiO2) and three NP sizes (1/3/5 nm diameter) with a representative lung surfactant membrane, and to study their binding kinetics. The calculated binding energies show that irrespective of size, Ag nanoparticles bind very strongly to the bilayer, while the NPs made of SiO2 or TiO2 experience very low to no binding. The unbiased simulations provide insight into how the NPs and membrane affect each other in terms of the solvent-accessible surface area (SASA) of the NPs and the defect types and fluidity of the membrane. Using these systematic fine-grained results in coarsening procedures will pave the way for simulations considering NP sizes that are well beyond the membrane thickness, i.e. closer to experimental dimensions, for which different binding characteristics and more significant membrane remodelling are expected.

A multiscale coarse-grained model to predict the molecular architecture and drug transport properties of modified chitosan hydrogels.

Hydrogels constructed with functionalized polysaccharides are of interest in a multitude of applications, chiefly the design of therapeutic and regenerative formulations. Tailoring the chemical modification of polysaccharide-based hydrogels to achieve specific drug release properties involves the optimization of many tunable parameters, including (i) the type, degree (χ), and pattern of the functional groups, (ii) the water-polymer ratio, and (iii) the drug payload. To guide the design of modified polysaccharide hydrogels for drug release, we have developed a computational toolbox that predicts the structure and physicochemical properties of acylated chitosan chains, and their impact on the transport of drug molecules. Herein, we present a multiscale coarse-grained model to investigate the structure of networks of chitosan chains modified with acetyl, butanoyl, or heptanoyl moieties, as well as the diffusion of drugs doxorubicin (Dox) and gemcitabine (Gem) through the resulting networks. The model predicts the formation of different network structures, in particular the hydrophobically-driven transition from a uniform to a cluster/channel morphology and the formation of fibers of chitin chains. The model also describes the impact of structural and physicochemical properties on drug transport, which was confirmed experimentally by measuring Dox and Gem diffusion through an ensemble of modified chitosan hydrogels.

Systematic Hydrogen-Bond Manipulations To Establish Polysaccharide Structure-Property Correlations.

A dense hydrogen-bond network is responsible for the mechanical and structural properties of polysaccharides. Random derivatization alters the properties of the bulk material by disrupting the hydrogen bonds, but obstructs detailed structure-function correlations. We have prepared well-defined unnatural oligosaccharides including methylated, deoxygenated, deoxyfluorinated, as well as carboxymethylated cellulose and chitin analogues with full control over the degree and pattern of substitution. Molecular dynamics simulations and crystallographic analysis show how distinct hydrogen-bond modifications drastically affect the solubility, aggregation behavior, and crystallinity of carbohydrate materials. This systematic approach to establishing detailed structure-property correlations will guide the synthesis of novel, tailor-made carbohydrate materials.

Tailoring the Chemical Modification of Chitosan Hydrogels to Fine-Tune the Release of a Synergistic Combination of Chemotherapeutics.

Combination chemotherapy with a defined ratio and sequence of drug release is a clinically established and effective route to treat advanced solid tumors. In this context, a growing body of literature demonstrates the potential of hydrogels constructed with chemically modified polysaccharides as depots for controlled release of chemotherapeutics. Identifying the appropriate modification in terms of physicochemical properties of the functional group and its degree of substitution (χ) to achieve the desired release profile for multiple drugs is, however, a complex multivariate problem. To address this issue, we have developed a computational toolbox that models the migration of a drug pair through a hydrated network of polysaccharide chains modified with hydrophobic moieties. In this study, we chose doxorubicin (DOX) and Gemcitabine (GEM) as model drugs, as their synergistic effect against breast cancer has been thoroughly investigated, and chitosan as the model polymer. Our model describes how the modification of chitosan chains with acetyl, butanoyl, and heptanoyl moieties at different values χ governs both the structure of the hydrogel network and drug migration through it. Our experimental data confirm the in silico predictions for both single- and dual-drug release and, most notably, the counterintuitive inversion of release vs χ that occurs when switching from a single- to a dual-drug system. Consensus between predicted and experimental data indicates that acetyl modifications (χ = 32-42%) and butanoyl modifications (χ = 19-24%) provide synergistic GEM/DOX release molar ratios (i.e., 5-10). Collectively, these results demonstrate the potential of this model in guiding the design of chemotherapeutic hydrogels to combat cancer.

Exploring triazine and heptazine based self assembled molecular materials through first principles investigations.

Two-dimensional materials formed from the molecular self assembly of monomers through noncovalent interactions are of great importance in designing complex nanostructures with desired properties. The carbon nitride based heterocyclic systems, triazine and heptazine, are found to be promising candidates for generating various self assembled materials through (N....H) hydrogen bonding. Here, we explored graphyne and graphdiyne-like self assembled structures for carbon nitride materials using the density functional theory calculations. We systematically investigated the monolayer structures, stacked structures in different configurations, as well as the surface assembled structures on the Au(111) surface. In all four different monolayer structures, the monomers interact through the N...H hydrogen bonding. The electronic structure results indicate that the electronic properties in these structures can be tuned through the variation in the length of the acetylinic unit. The minimum energy stacked bilayer structure of triazine based material exactly matches with the experimentally reported structure. Surface assembled studies of the triazine based system show strong interaction between the Au(111) surface and the carbon nitride monolayer. Graphical abstract Self assembled two-dimensional molecular materials as well as the surface assemblies of triazine and heptazine based precursors are computationally investigated.

Perception of Suicide Risk in Mental Health Professionals.

This study employed an independent-groups design (4 conditions) to investigate possible biases in the suicide risk perception of mental health professionals. Four hundred participants comprising doctors, nurses and social workers viewed a vignette describing a fictitious patient with a long-term mental illness. The case was presented as being drawn from a sample of twenty similar clinical case reports, of which 10 were associated with an outcome of suicide. The participant tasks were (i) to decide whether the presented vignette was one of those cases or not, and (ii) to provide an assessment of confidence in that decision. The 4 conditions were used to investigate whether the presence of an associated face, and the nature of the emotional state expressed by that face, affected the response profile. In fact, there were no significant differences between conditions, but there was a significant bias across all conditions towards associating the vignette with suicide, despite the base rate being pre-determined at 50%. The bias was more pronounced in doctors and in male respondents. Moreover, many participants indicated substantial confidence in their decisions. The results are discussed in terms of availability bias and over-confidence bias.

High specificity of the medical symptom validity test in patients with very severe memory impairment.

Failure on effort tests usually implies insufficient effort to produce valid cognitive test scores. However, many people with very severe cognitive impairment, such as dementia patients, will produce failing scores on nearly all effort tests. In such patients, effort tests have low specificity. The Medical Symptom Validity Test (MSVT) and the nonverbal MSVT (NV-MSVT) were designed to address this problem. They produce profiles of scores across multiple subtests to facilitate discrimination between low scores from people trying to feign impairment and low scores attributable to severe impairment. To study the specificity of the MSVT and NV-MSVT in people with very severe memory impairment, we tested (a) 10 institutionalized patients with dementia and (b) 10 volunteers who were asked to simulate memory impairment. It was hypothesized that the "possible dementia profile" would be found significantly more often in the dementia patients than in the simulators. The MSVT and the NV-MSVT both displayed 100% specificity in the dementia group, while retaining a combined sensitivity of 80% to suboptimal effort in the simulator group.

Effect of buprenorphine on psychomotor functions in patients on buprenorphine maintenance.

OBJECTIVE: Patients on buprenorphine maintenance for opioid dependence often abuse its additional doses over and above the maintenance dose. Being a psychoactive agent, it may affect psychomotor performance with all its consequences, for example, effect on quality of life. This study was conducted to assess the effects of its additional doses on psychomotor performance in patients who are maintained on it. DESIGN AND SETTING: This was an interventional study, carried out in an in-patient setting in a tertiary care national drug dependence treatment center. PARTICIPANTS: It included 19 subjects maintained on buprenorphine, 4 mg/d (s/L) for at least a month. INTERVENTION: Maintenance dose was followed by three administrations of buprenorphine, 2 mg, at two hourly intervals (cumulative dose design). MAIN OUTCOME MEASURES: Subjects were assessed on digit symbol substitution test, trail making, digit span, and delayed recall, after each administration and the next morning. RESULTS: Performance of subjects on Digit Symbol Substitution Test (chi2 = 52.98, p < 0.000) and Trail Making Test-A (chi2 = 26.29, p < 0.000) and B (chi2 = 42.08, p < 0.000) improved significantly with each assessment while other tests were unaffected. CONCLUSIONS: Improvement in psychomotor performance (which could be true effect of drug itself or a result of other factors, eg, inadequate maintenance dose or practice effect) though contrasting with some of the earlier findings, does have significant clinical implications regarding the long-term use of buprenorphine. It would be worthwhile repeating this type of study in a placebo controlled design to further verify the results.