Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

α-Synuclein kinetically regulates the nascent fusion pore dynamics.

α-Synuclein (α-synFL) is central to the pathogenesis of Parkinson's disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-synFL's role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-synFL's role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-SynFL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-synFL's inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-synFL's ability to alter fusion pore dynamics. α-SynFL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF-resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-synFL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca2+•syt1 triggered α-synFL-resistant SNARE complex formation that rendered α-synFL inactive in modulating pore properties. This study reveals a key role of α-synFL in controlling vesicular secretion.

Perception and Acceptance of the Newly Introduced COVID-19 Precaution (THIRD) Vaccination Dose among Health Workers at an Indian Medical College.

INTRODUCTION: India has run a nationwide vaccination campaign against COVID-19, which has recently introduced a precaution (third) dose for health workers. This study assessed the perception and attitude of health workers toward the Indian vaccination campaign against COVID-19, with an emphasis on this major change. MATERIALS AND METHODS: A printed questionnaire was distributed among health-care workers at the Medical College of West Bengal. The completed forms were analyzed. RESULTS: Most of the participants were doctors (83.7%). Although all had received two doses of vaccine before, 44.4% were unwilling to be vaccinated with the third dose in the present scenario. The majority (63.8%) of the patients were concerned about side effects. The emergence of new COVID strains (65.6%) was viewed as a threat to the effectiveness of the vaccines. Participants with higher age, comorbidities, and those with the perception that the third dose was being introduced appropriately and would be effective against newer strains of the vaccine tended to be more willing to get vaccinated with the precaution dose compared to their counterparts. CONCLUSION: A dilemma regarding the acceptance of precaution doses was noted among health workers. This warrants the availability of more comprehensive information to increase acceptance of these vaccines.

Enzymatic dispersion of biofilms: An emerging biocatalytic avenue to combat biofilm-mediated microbial infections.

Drug resistance by pathogenic microbes has emerged as a matter of great concern to mankind. Microorganisms such as bacteria and fungi employ multiple defense mechanisms against drugs and the host immune system. A major line of microbial defense is the biofilm, which comprises extracellular polymeric substances that are produced by the population of microorganisms. Around 80% of chronic bacterial infections are associated with biofilms. The presence of biofilms can increase the necessity of doses of certain antibiotics up to 1000-fold to combat infection. Thus, there is an urgent need for strategies to eradicate biofilms. Although a few physicochemical methods have been developed to prevent and treat biofilms, these methods have poor efficacy and biocompatibility. In this review, we discuss the existing strategies to combat biofilms and their challenges. Subsequently, we spotlight the potential of enzymes, in particular, polysaccharide degrading enzymes, for biofilm dispersion, which might lead to facile antimicrobial treatment of biofilm-associated infections.

Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase.

Early detection of Alzheimer's disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the "acetylcholine-mimic" approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.

Use of cardiopulmonary bypass machine in intensive care unit as a short term mechanical circulatory support for recovery of cardiac function.

INTRODUCTION: Extracorporeal Membrane Oxygenation (ECMO) is used as a bridge to recovery of cardiac function following completion of congenital cardiac surgeries where there is failure to wean from cardiopulmonary bypass (CPB) or severe low cardiac output states in the post operative periods. Although ECMO is a well-established form of mechanical circulatory support, the associated cost can be a huge financial burden on families. We are an ECMO center and use the same in post operative congenital cardiac surgeries for mechanical cardiovascular support if needed. However, a significant proportion of the children, whom we operate, are funded by government aides. The resources are limited in such circumstances. If needed, we use the same CPB circuit and cannulae used in the Operating Room (OR) and support them at a significantly lower cost compared to ECMO. METHODS: We report our experience of using conventional CPB machine as a short-term bridge to recovery of cardiac function in Intensive Care Unit where there was limitation of funds. Essentially same CPB circuit with roller pump is retained, by omitting cardiotomy suckers. We use D901 Lilliput 1 Oxygenator (Sorin, Italy) for children <5 kg and D902 Lilliput 2 (Sorin, Italy) Oxygenator for children >5 Kg. RESULTS: We supported nine patients on CPB between March 2019 and December 2021. During this time, 1392 congenital cardiac surgeries were performed. We could wean off three patients (33.3%) and discharge two patients (22.2%). Our support time ranged from 21 h to 60 h with a median of 48 h. Beyond 48 h of support, we experienced several CPB induced complications in our cohort. CONCLUSION: In resource-limited settings, conventional CPB machines can be used for short-term cardiac support. Although results may not be comparable to using ECMO, some patients can be definitely salvaged, who would otherwise die in the absence of institution of mechanical circulatory support.

Morbidity pattern and health-care seeking among women domestic workers: Perspective from an Indian metropolis.

Background: Domestic work being unrecognized as a formal form of occupation in India, workers are often deprived of basic social security, including access to proper health care. Self-negligence coupled with societal neglect makes them vulnerable to injury and a variety of illnesses. Objectives: We aimed to study the morbidity pattern of women domestic workers residing in a slum area of Kolkata, West Bengal, India. Materials and Methods: An observational descriptive cross-sectional study was carried out by interviewing and clinically examining 106 randomly selected women domestic workers. Results: The majority (82.08%) had health complaints: heart burn being most common (40.57%). Musculoskeletal and dermatological issues were common. Anemia (31.25%) was a common prediagnosed morbidity. Pallor (33.96%) and dental caries (31.13%) were common; 50.94% reported workplace injury. Of those having complaints, 77.36% sought health care, 59.43% of whom relied on health facilities. Lack of time (54.05%), felt need (35.14%) and money (21.62%) affected adequate care seeking. Participants with cardiorespiratory complaints tended to visit health facilities significantly more (P < 0.05). Conclusion: Health vulnerabilities and neglectful behavior among domestic workers are evident, making the need to sensitize them about their health risks and ways to overcome such issues very vital. Awareness should also be generated about government health schemes to encourage timely health checkup and necessary intervention.

Biochemical Investigation of Membrane-Bound Cytochrome b5 and the Catalytic Domain of Cytochrome b5 Reductase from Arabidopsis thaliana.

The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene-maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.

Factors predicting proper handwashing practice amidst the COVID-19 pandemic in India: a field-based cross-sectional study.

Proper handwashing is one of the effective ways to prevent many communicable diseases, including COVID-19. We explored the handwashing practices in a rural Indian population before the probable third wave of the COVID-19 pandemic. A data collection schedule was administered to eligible adult members of a rural community, selected by multi-stage sampling, to assess their pattern and practice of handwashing. All 176 respondents washed their hands after defecation, 82.4 and 80.7% washed hands after urination and before taking food, respectively, while 68.2% of respondents washed hands after coming back from outdoors. Among those who handwashed, 82.9% used soap water after defecation; 46.2, 45.8 and 50.8% washed hands with soap water after urination, before taking food and after visiting outdoors, respectively. Only a quarter (24.4%) of all the participants used soap water for handwashing consistently after defecation, after urination, before taking food and after coming home. The more educated, those coming from higher socioeconomic stratum and working from home, were more likely to report proper handwashing practice. Handwashing, as recommended by health agencies, for restraining COVID-19 infection, was not noticed in the majority of the participants. Better awareness of handwashing is recommended to help restrain COVID-19 in the Indian population.

Basics of extra corporeal membrane oxygenation: a pediatric intensivist's perspective.

Extra Corporeal membrane oxygenation (ECMO) is one of the most advanced forms of life support therapy in the Intensive Care Unit. It relies on the principle where an external artificial circuit carries venous blood from the patient to a gas exchange device (oxygenator) within which blood becomes enriched with oxygen and has carbon dioxide removed. The blood is then returned to the patient via a central vein or an artery. The goal of ECMO is to provide a physiologic milieu for recovery in refractory cardiac/respiratory failure. The technology is not a definitive treatment for a disease, but provides valuable time for the body to recover. In that way it can be compared to a bridge, where patients are initiated on ECMO as a bridge to recovery, bridge to decision making, bridge to transplant or bridge to diagnosis. The use of this modality in children is not backed by a lot of randomized controlled trials, but the use has increased dramatically in our country in last 10 years. This article is not intended to provide an in-depth overview of ECMO, but outlines the basic principles that a pediatric intensive care physician should know in order to manage a kid on ECMO support.

Cannabis use in patients with early psychosis is associated with alterations in putamen and thalamic shape.

Around half of patients with early psychosis have a history of cannabis use. We aimed to determine if there are neurobiological differences in these the subgroups of persons with psychosis with and without a history of cannabis use. We expected to see regional deflations in hippocampus as a neurotoxic effect and regional inflations in striatal regions implicated in addictive processes. Volumetric, T1w MRIs were acquired from people with a diagnosis psychosis with (PwP + C = 28) or without (PwP - C = 26) a history of cannabis use; and Controls with (C + C = 16) or without (C - C = 22) cannabis use. We undertook vertex-based shape analysis of the brainstem, amygdala, hippocampus, globus pallidus, nucleus accumbens, caudate, putamen, thalamus using FSL FIRST. Clusters were defined through Threshold Free Cluster Enhancement and Family Wise Error was set at p < .05. We adjusted analyses for age, sex, tobacco and alcohol use. The putamen (bilaterally) and the right thalamus showed regional enlargement in PwP + C versus PwP - C. There were no areas of regional deflation. There were no significant differences between C + C and C - C. Cannabis use in participants with psychosis is associated with morphological alterations in subcortical structures. Putamen and thalamic enlargement may be related to compulsivity in patients with a history of cannabis use.

Membrane association of monotopic phosphoglycosyl transferase underpins function.

Polyprenol phosphate phosphoglycosyl transferases (PGTs) catalyze the first membrane-committed step in assembly of essential glycoconjugates. Currently there is no structure-function information to describe how monotopic PGTs coordinate the reaction between membrane-embedded and soluble substrates. We describe the structure and mode of membrane association of PglC, a PGT from Campylobacter concisus. The structure reveals a unique architecture, provides mechanistic insight and identifies ligand-binding determinants for PglC and the monotopic PGT superfamily.

Naked Eye Cd2+ Ion Detection and Reversible Iodine Uptake by a Three-Dimensional Pillared-Layered Zn-MOF.

A novel metal-organic framework (MOF), [Zn2(tdca)2(bppd)2]·2DMF, has been synthesized solvothermally using the ligand thiophene-2,5-dicarboxylic acid (H2tdca), coligand N,N'-bis(4-pyridylmethylene)-1,4-benzenediamine (bppd), and Zn(NO3)2. Single crystal X-ray crystallography reveals that the titled MOF is a three-dimensional pillared-layered MOF. A layer is constituted by a Zn(tdca) unit, and the layers are stabilized by the long hydrocarbon coligand, bppd, which acts as a pillar. A rectangular pore size of 11.42 × 8.12 Å2 is found in the framework. The porous framework is found to be an excellent fluorescence sensor for the detection of toxic Cd2+ ion. The sensor shows high selectivity and sensitivity and a quick response toward Cd2+. The synthesized MOF is able to not only detect cadmium ions but also adsorb iodine in the gas phase. The MOF can adsorb ∼66% iodine, verified by thiosulfate-iodine titration and TG analysis. Adsorbed iodine can also be removed easily in acetonitrile as well as in n-hexane, which shows that iodine can be reversibly loaded as well as unloaded into the framework.

A Chemodosimetric Approach for Fluorimetric Detection of Hg2+ Ions by Trinuclear Zn(II)/Cd(II) Schiff Base Complex: First Case of Intermediate Trapping in a Chemodosimetric Approach.

The present work discloses the application of two fluorescent zinc and cadmium complexes (1 and 2) for sensing of Hg(II) ions through a chemodosimetric approach. The ligand under consideration in this work is a N2O donor Schiff base ligand (E)-4-bromo-2-(((2-morpholinoethyl)imino)methyl)phenol (HL), which has been harnessed to generate complexes [Zn3L2(OAc)4] (1) and [Cd3L2(OAc)4] (2). X-ray single crystal diffraction studies unveil the trinuclear skeleton of complexes 1 and 2. Both complexes have been found to be highly fluorescent in nature. However, the quantum efficiency of Zn(II) complex (1) dominates over the Cd(II) analogue (2). The absorption and emission spectroscopic properties of the complexes have been investigated by density functional theory. Complexes 1 and 2 can detect Hg2+ ions selectively by fluorescence quenching, and it is noteworthy to mention that the mechanism of sensing is unique as well as interesting. In the presence of Hg2+ ions, complexes 1 and 2 are transformed to mononuclear mercuric intermediate complex (3) and finally to a trinuclear mercuric complex (4) by hydrolysis. We have successfully trapped the intermediate complex 3, and we characterized it with the aid of X-ray crystallography. Transformation of complexes 1 and 2 to intermediate complex 3 and finally to 4 has been established by UV-vis spectroscopy, fluorescence spectroscopy, ESI-MS spectroscopy, 1H NMR spectroscopy, and X-ray crystallography. The spontaneity of the above conversion is well supported by thermodynamic aspects as reflected from density functional theoretical calculations.

In vivo efficacy studies of novel quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models.

Lung cancer is the most common cancer and leading cause of cancer-related deaths worldwide. The first-generation reversible, ATP-competitive inhibitors gefetinib and elotinib showed good clinical responses in lung adenocarcinoma tumors (NSCLC). But almost all patients developed resistance to these inhibitors over time. Such resistance of EGFR inhibitors was frequently linked to the acquired L858R and T790M point mutations in the kinase domain of EGFR. To overcome these resistance problems, the second and the third generation inhibitors have been discovered. FDA approved afatinib, the second generation irreversible inhibitor and osimitinib, the third generation irreversible EGFR inhibitors for the treatments of NSCLC. We identified new covalent quinazoline inhibitors (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (6d) and (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethyl-amino)but-2-enamide (6h) that exhibited potent EGFR kinase inhibitory activities on L858R and T790M mutations. The compound 6 h showed selectivity similar to AZD9291 (osimertinib) in mutated and wild type tumor cell lines. In vitro cell assay 6d and 6h were better than afatinib and osimertinib. In vivo antitumor efficacy studies of these compounds were done in NCI-H1975 mice xenografts.

Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate.

Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs.

Exploring aggregation-induced emission through tuning of ligand structure for picomolar detection of pyrene.

Tuning of ligand structures through controlled variation of ring number in fused-ring aromatic moiety appended to antipyrine allows detection of 7.8 × 10-12  M pyrene via aggregation-induced emission (AIE) associated with 101-fold fluorescence enhancement. In one case, antipyrine unit is replaced by pyridine to derive bis-methylanthracenyl picolyl amine. The structures of four molecules have been confirmed by single crystal X-ray diffraction analysis. Among them, pyrene-antipyrine conjugate (L) undergoes pyrene triggered inhibition of photo-induced electron transfer (PET) leading to water-assisted AIE.

Design, synthesis of orally bioavailable novel anaplastic lymphoma kinase (ALK) inhibitor diphenylaminopyrimidine analogs and efficacy study on NCI-H2228 xenografts mice model.

Anaplastic lymphoma kinase (ALK) is an attractive therapeutic target for the treatment of non-small cell lung cancer (NSCLC). Within our ALK drug discovery program, we identified novel deuterated 2,4-diarylamino pyrimidine compounds as potent ALK inhibitors. The compound 11 showed better in vitro activity and in vivo efficacy with good pharmacokinetic profile. In vivo efficacy of compound 11 was better than standard drug ceritinib in NCI-H2228 xenograft mice model.

Discovery of new quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors and their anticancer activities - Part 1.

Overexpression of EGFR and HER2 are observed in many breast, ovarian, colon and prostate cancers. The second and third generation irreversible EGFR/HER2 dual kinase inhibitors became popular after the approval of Afatinib by FDA to overcome the mutation related problem. To find efficacious drug candidates, a series of novel quinazoline derivatives were designed, synthesized and evaluated as dual EGFR/HER2 tyrosine kinase (TK) inhibitors. Selected twenty four compounds were reported here with significant inhibitory activities against EGFR/HER2 tyrosine kinases. Several compounds showed nanomolar IC50 values. In vitro studies of quinazoline derivatives were done on NCI-H1975, HCC827, A431, MDA MB-453 cell lines. The compounds 1a, 1d and 1v were found more potent compared to standard drug afatinib. In vivo efficacy study of 1d on nude mice NCI-H1975 tumour xenograft model was discussed.

Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes.

Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins-protective antigen (PA), lethal factor (LF), and edema factor-translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity.