SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

Molecular Mobility-Mediated Regulation of E-Cadherin Adhesion.

Cells in epithelial tissues utilize homotypic E-cadherin interaction-mediated adhesions to both physically adhere to each other and sense the physical properties of their microenvironment, such as the presence of other cells in close vicinity or an alteration in the mechanical tension of the tissue. These position E-cadherin centrally in organogenesis and other processes, and its function is therefore tightly regulated through a variety of means including endocytosis and gene expression. How does membrane molecular mobility of E-cadherin, and thus membrane physical properties and associated actin cytoskeleton, impinges on the assembly of adhesive clusters and signaling is discussed.

Swarm Intelligence in Internet of Medical Things: A Review.

Continuous advancements of technologies such as machine-to-machine interactions and big data analysis have led to the internet of things (IoT) making information sharing and smart decision-making possible using everyday devices. On the other hand, swarm intelligence (SI) algorithms seek to establish constructive interaction among agents regardless of their intelligence level. In SI algorithms, multiple individuals run simultaneously and possibly in a cooperative manner to address complex nonlinear problems. In this paper, the application of SI algorithms in IoT is investigated with a special focus on the internet of medical things (IoMT). The role of wearable devices in IoMT is briefly reviewed. Existing works on applications of SI in addressing IoMT problems are discussed. Possible problems include disease prediction, data encryption, missing values prediction, resource allocation, network routing, and hardware failure management. Finally, research perspectives and future trends are outlined.

Competition for shared downstream signaling molecules establishes indirect negative feedback between EGFR and EphA2.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.

Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility.

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.

Intracellular Ionic Strength Sensing Using NanoLuc.

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen-NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

Fabrication of Multicomponent, Spatially Segregated DNA and Protein-Functionalized Supported Membrane Microarray.

Deoxyribonucleic acid (DNA) has been used as a material for a variety of applications, including surface functionalization for cell biological or in vitro reconstitution studies. Use of DNA-based surface functionalization eliminates limitations of multiplexing posed by traditionally used methods in applications requiring spatially segregated surface functionalization. Recently, we have reported a stochastic, membrane fusion-based strategy to fabricate multicomponent membrane array substrates displaying spatially segregated protein ligands using biotin-streptavidin and Ni-NTA-polyhistidine interactions. Here, we report the delivery of DNA oligonucleotide-conjugated lipid molecules to membrane corrals, allowing spatially segregated membrane corral functionalization in a membrane microarray. Incubation of microbeads coated with the supported membrane resulted in an exchange of lipid contents with planar membrane corrals present on a micropatterned substrate. Increases in the system temperature and membrane corral size resulted in alterations in the rate constant of lipid exchange, which are in agreement with our previously developed analytical model and further confirm that lipid exchange is a diffusion-based process that takes place after the formation of a long "fusion-stalk" between the two membranes. We take advantage of the physical dimensions of the fusion-stalk with a large aspect ratio to deliver DNA oligonucleotide-conjugated lipid molecules to membrane corrals. We believe that the ability to functionalize membrane corrals with DNA oligonucleotides significantly increases the utility of the stochastic fusion-mediated lipid delivery strategy in the functionalization of biomolecules such as DNA or DNA-conjugated protein ligands.

Interfacial Forces Dictate the Pathway of Phospholipid Vesicle Adsorption onto Silicon Dioxide Surfaces.

The pathway of vesicle adsorption onto a solid support depends on the material composition of the underlying support, and there is significant interest in developing material-independent strategies to modulate the spectrum of vesicle-substrate interactions on a particular surface. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we systematically investigated how solution pH and membrane surface charge affect vesicle adsorption onto a silicon dioxide surface. While vesicle adsorption and spontaneous rupture to form complete supported lipid bilayer (SLBs) occurred in acidic conditions, it was discovered that a wide range of adsorption pathways occurred in alkaline conditions, including (i) vesicle adsorption and spontaneous rupture to form complete SLBs, (ii) vesicle adsorption and spontaneous rupture to form incomplete SLBs, (iii) irreversible adsorption of intact vesicles, (iv) reversible adsorption of intact vesicles, and (v) negligible adsorption. In general, SLB formation became more favorable with increasingly positive membrane surface charge although there were certain conditions at which attractive electrostatic forces were insufficient to promote vesicle rupture. To rationalize these findings, we discuss how solution pH and membrane surface charge affect interfacial forces involved in vesicle-substrate interactions. Taken together, our findings present a comprehensive picture of how interfacial forces dictate the pathway of phospholipid vesicle adsorption onto silicon dioxide surfaces and offer a broadly applicable framework to characterize the interactions between phospholipid vesicles and inorganic material surfaces.

Membrane Reconstitution of Monoamine Oxidase Enzymes on Supported Lipid Bilayers.

Monoamine oxidase A and B (MAO-A and B) are mitochondrial outer membrane enzymes that are implicated in a number of human diseases, and the pharmacological inhibition of these enzymes is a promising therapeutic strategy to alleviate disease symptoms. It has been suggested that optimal levels of enzymatic activity occur in the membrane-associated state, although details of the membrane association process remain to be understood. Herein, we have developed a supported lipid bilayer platform to study MAO-A and B binding and evaluate the effects of known pharmacological inhibitors on the membrane association process. By utilizing the quartz crystal microbalance-dissipation (QCM-D) technique, it was determined that both MAOs exhibit tight binding to negatively and positively charged bilayers with distinct concentration-dependent binding profiles while only transiently binding to neutral bilayers. Importantly, in the presence of known inhibitors, the MAOs showed increased binding to negatively charged bilayers, although there was no effect of inhibitor treatment on binding to positively charged bilayers. Taken together, our findings establish that the membrane association of MAOs is highly dependent on membrane surface charge, and we outline an experimental platform to support the in vitro reconstitution of monoamine oxidases on synthetic membranes, including the evaluation of pharmacological drug candidates.

Early events in the assembly of E-cadherin adhesions.

E-cadherin is a calcium dependent cell adhesion molecule that is key to the organization of cells in the epithelial tissue. It is a multidomain, trans-membrane protein in which the extracellular domain forms the homotypic, adhesive interaction while the intracellular domain interacts with the actin cytoskeleton through the catenin family of adaptor proteins. A number of recent studies have provided novel insights into the mechanism of adhesion formation by this class of adhesion proteins. Here, we describe an updated view of the process of E-cadherin adhesion formation with an emphasis on the role of molecular mobility, clustering, and active cellular processes.

E-cadherin junction formation involves an active kinetic nucleation process.

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

Sustained α-catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force.

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

A Microbead Supported Membrane-Based Fluorescence Imaging Assay Reveals Intermembrane Receptor-Ligand Complex Dimension with Nanometer Precision.

Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor-ligand dimension at adhesions established by receptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epi-fluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required.

Physical activity levels in Bangladeshi adults: results from STEPS survey 2010.

OBJECTIVES: Physical inactivity is an established risk factor for non-communicable diseases (NCD) and identified as the major public health concern worldwide. However, nationally representative and internationally comparable data on physical activity (PA) are lacking in Bangladesh. The objective of this paper was to determine nationally representative prevalence of PA levels among Bangladeshi adults. STUDY DESIGN: Cross-sectional survey. METHODS: Data, on PA for this paper, were analysed from the NCD risk factors survey 2010 in Bangladesh. A standardized approach known as STEPS (STEPSwise approach to Surveillance for NCD risk factors) was followed for this survey. A total of 9275 adults (aged ≥ 25 years) were interviewed. Data on PA were processed and analysed according to Global Physical Activity Questionnaire (GPAQ) version 2 analysis framework. RESULTS: Of total 9275 respondents 4312 were men and 4963 women with a mean age of 42.4 (±13.5) years. Median MET-minutes of total PA in a typical week was double in rural areas (3360) than urban (1680) areas. The overall country wide prevalence of low PA was 34.5% (95% confidence interval, 33.5-35.5), urban 37.7% (36.3-39.1) and rural 31.6% (30.3-32.9). Women in general were more inactive (women, 53.6% [52.2-55.0], men 15.4% [14.9-17.1]). The main contributions to total PA were from work (urban 47.0%, rural 61.0%), and active commuting (38.0%, 30.0%) domains. Leisure-time PA represented only a small proportion (15.0%, 9.0%). CONCLUSIONS: Insufficient physical activity is highly prevalent among the Bangladeshi adult population. Promoting overall PA at leisure-time and commuting considering country context can be feasible options with special attention to the women.

A report of heat stroke in two Nigerian siblings.

Infants and children are at higher risk of heat stroke for several reasons. We report these cases to highlight the danger of leaving children unsupervised in vehicles, aid prompt diagnosis, and management of heat stroke. Two Nigerian siblings aged ranges 5 and 3 years old, were trapped inside an unlocked vehicle and subsequently developed heat stroke. Both children presented with hyperthermia, severe dehydration, convulsions, and loss of consciousness. One of them also had hematuria. They were treated by spraying water onto their bodies to bring down the temperature, intravenous fluid resuscitation, oxygen therapy, and anticonvulsants. Both eventually recovered and were discharged with no obvious neurologic sequalae, but are being followed-up.

Comparison of efficacy of labetalol and fentanyl for attenuating reflex responses to laryngoscopy and intubation.

Stress response due to laryngoscopy and intubation has been universally recognized phenomenon resulting in increase in heart rate, arterial, intracranial, and intraocular pressure. Various pharmacological approaches have been used to blunt or attenuate such pressure responses. This prospective, randomized, placebo controlled, double blinded study was designed to compare the efficacy of bolus dose of Labetalol and Fentanyl for attenuating reflex responses to laryngoscopy and intubation. Ninety patients with physical status of ASA I and II were scheduled for elective surgery under standard protocol of general anaesthesia, randomly allocated into three groups, consisting of 30 patients in each group, assigned as C (Control), L (Labetalol), and F (Fentanyl). In control group 10ml of 0.9% saline, in Labetalol group 0.25 mg/kg Labetalol and in Fentanyl group 2µgm/kg of Fentanyl were given intravenously at 3 minutes prior to laryngoscopy and intubation. Pulse rate, systolic, diastolic, mean arterial pressure and rate pressure products (RPP) were recorded before and after premedication, after administration of study drugs and at 1, 3, 5, 10 and 15 minutes after intubation. For statistical analysis of data, ANOVA tests were performed for comparison between groups. There were an increase in heart rate, systolic, diastolic, mean arterial pressures and rate pressure product in all the three groups after intubation in comparison to base line value. But the rise was minimum in L and F group as compared to C group which is statistically significant and also minimum in L group as compared to F group. So Labetalol is better agent for attenuation of laryngoscopic and intubation reflex.

Distribution and Characteristics of Congenital Cardiac Surgery Centers within Bangladesh.

Congenital heart disease is a leading cause of non-communicable childhood death. This is especially true in nations with limited resources where shortages of skilled workforce, healthcare facilities, and essential equipment limit the ability to provide care. This retrospective study was designed to determine the volume and distribution of surgical care being provided to patients with congenital heart disease in Bangladesh, as well as to characterize the facilities providing such care. Pre-existing survey data of hospitals performing congenital heart surgery in the year 2022 in Bangladesh was obtained. Additional information was gathered on these facilities, including hospital location and type. The distribution of care by geographic location, type of facility, and volume of cases was reported. Overall, a total of 2333 surgeries were performed in 2022 at 28 facilities. The majority of hospitals were performing <50 cases per year, while a small number (5) provided greater than 50.0% of all surgeries. In addition, while the majority of hospitals were private in nature, the majority of surgeries occurred at not-for-profit hospitals. There was a large geographic skew of surgeries and hospitals being located within the city of Dhaka (79.0% of centers and 94.0% of surgeries). The data suggests that, though there has been great progress in increasing the number of surgeries performed in Bangladesh, the vast majority of patients still do not have access to care. In addition, nearly all care is being provided in Dhaka, which presents challenges for patients who come from across the nation seeking care. Finally, there is a great need for further research to fully understand the challenges faced and find workable solutions.

BRET-based biosensors for SARS-CoV-2 oligonucleotide detection.

The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype-the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.

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

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

Haemodynamic and end tidal CO2 changes during laparoscopic cholecystectomy under general anaesthesia.

A prospective observational study was done on 50 patients to investigate the haemodynamic and end tidal CO2 (EtCO2) changes in healthy patients without cardiopulmonary pathology during elective laparoscopic cholecystectomy in head up position under standard protocol of general anaesthesia. During surgery, intra abdominal pressure was maintained at 15 mmHg by a CO2 insufflator and minute ventilation was controlled with a constant tidal volume and fixed respiratory rate. Haemodynamic parameters, EtCO2, SpO2 and ECG were recorded before and after induction and positioning of the patients and at 5 minutes interval for the first 30 minutes, then 10 minutes interval for the rest of the period. Highly significant increase (p<0.001) in pulse rate, systolic, diastolic and mean arterial pressure occurred at 30 minutes after insufflations and positioning of the patient. A very highly significant (p<0.001) increase in EtCO2 from the base line was at 40 minutes after insufflations and positioning of the patients. There was no change in SpO2 and ECG. This study supports the significant physiological changes in terms of haemodynamic and EtCO2 during laparoscopic cholecystectomy and recommends the meticulous monitoring of these parameters during the surgery and balance the benefit of laparoscopy against the intra operative risk.