Klebsiella pneumoniae carbapenemase variant 44 acquires ceftazidime-avibactam resistance by altering the conformation of active-site loops.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all ß-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI-resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino acid duplication in one of the active-site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency toward imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, than the WT KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower AVI carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates AVI carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.

Mutagenesis and structural analysis reveal the CTX-M ß-lactamase active site is optimized for cephalosporin catalysis and drug resistance.

CTX-M ß-lactamases are a widespread source of resistance to ß-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 ß-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A ß-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra.

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

Structural Insights into Dihydroxylation of Terephthalate, a Product of Polyethylene Terephthalate Degradation.

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis-diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDOKF1). TPDOKF1 exhibited substrate specificity for TPA (kcat/Km = 57 ± 9 mM-1 s-1). The TPDOKF1 structure harbors characteristic RO features as well as a unique catalytic domain that rationalizes the enzyme's function. The docking and mutagenesis studies reveal that its substrate specificity for TPA is mediated by the Arg309 and Arg390 residues, positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, as its mutation to alanine decreases the activity (kcat) by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. IMPORTANCE Global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential for tackling this. Microbial utilization of this released product, TPA, is an emerging and promising strategy for waste-to-value creation. Research in the last decade has identified terephthalate dioxygenase (TPDO) as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 µM-1s-1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Conformational flexibility enables catalysis of phthalate cis-4,5-dihydrodiol dehydrogenase.

Phthalate cis-4,5-dihydrodiol dehydrogenase (PhtC), the second enzyme of the phthalate catabolic pathway, catalyzes the dehydrogenation of cis-4,5-dihydrodiol phthalate (DDP). Here, we report the structural and biochemical characterization of PhtC from Comamonas testosteroni KF1 (PhtCKF1). With biochemical experiments, we have determined the enzyme's catalytic efficiency (kcat/Km) with DDP as 2.6 ± 0.5 M-1s-1, over 10-fold higher than with cis-3,4-dihydrodiol phthalate (CDP). To understand the structural basis of these reactions, the crystal structures of PhtCKF1 in apo-form, the binary complex with NAD+, and the ternary complex with NAD+ and 3-hydroxybenzoate (3HB) were determined. These crystal structures reveal that the binding of 3HB induces a conformational change in the substrate-binding loop. This conformational change causes the opening of the NAD + binding site while trapping the 3HB. The PhtCKF1 crystal structures show that the catalytic domain of PhtCKF1 is larger than that of other structurally characterized homologs and does not align with other cis-diol dehydrogenases. Structural and mutational analysis of the substrate-binding loop residues, Arg164 and Glu167 establish that conformational flexibility of this loop is necessary for positioning the substrate in a catalytically competent pose, as substitution of either of these residues to Ala did not yield the dehydrogenation activity. Further, based on the crystal structures of PhtCKF1 and related structural homologs, a reaction mechanism is proposed. Finally, with the biochemical analysis of a variant M251LPhtCKF1, the broader substrate specificity of this enzyme is explained.

Deciphering the enigma of missing DNA binding domain of LacI family transcription factors.

Catabolite repressor activator (Cra) is a member of the LacI family transcriptional regulator distributed across a wide range of bacteria and regulates the carbon metabolism and virulence gene expression. In numerous studies to crystallize the apo form of the LacI family transcription factor, the N-terminal domain (NTD), which functions as a DNA-binding domain, has been enigmatically missing from the final resolved structures. It was speculated that the NTD is disordered or unstable and gets cleaved during crystallization. Here, we have determined the crystal structure of Cra from Escherichia coli (EcCra). The structure revealed a well-defined electron density for the C-terminal domain (CTD). However, electron density was missing for the first 56 amino acids (NTD). Our data reveal for the first time that EcCra undergoes a spontaneous cleavage at the conserved Asn 50 (N50) site, which separates the N-terminal DNA binding domain from the C-terminal effector molecule binding domain. With the site-directed mutagenesis, we confirm the involvement of residue N50 in the spontaneous cleavage phenomenon. Furthermore, the Isothermal titration calorimetry (ITC) assay of the EcCra-NTD with DNA showed EcCra-NTD is in a functional conformation state and retains its DNA binding activity.

Structural and Biochemical Analyses Reveal that Chlorogenic Acid Inhibits the Shikimate Pathway.

Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with KD (equilibrium dissociation constant) values of 6.3 µM and 0.5 µM, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 ± 21 µM, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 µM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules.IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes.

Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

CONFORMATIONAL FLEXIBILITY IS A CRITICAL FACTOR IN DESIGNING BROAD-SPECTRUM HUMAN NOROVIRUS PROTEASE INHIBITORS.

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is, therefore, an excellent target for developing small molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases.

Biophysical and modeling-based approach for the identification of inhibitors against DOHH from Leishmania donovani.

The amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) occurs only in isoforms of eukaryotic translation factor 5A (eIF5A) and has a role in initiating protein translation. Hypusinated eIF5A promotes translation and modulates mitochondrial function and oxygen consumption rates. The hypusination of eIF5A involves two enzymes, deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH is the second enzyme that completes the synthesis of hypusine and the maturation of eIF5A. Our current study aims to identify inhibitors against DOHH from Leishmania donovani (LdDOHH), an intracellular protozoan parasite causing Leishmaniasis in humans. The LdDOHH protein was produced heterologously in Escherichia coli BL21(DE3) cells and characterized biochemically. The three-dimensional structure was predicted, and the compounds folic acid, scutellarin and homoarbutin were selected as top hits in virtual screening. These compounds were observed to bind in the active site of LdDOHH stabilizing the structure by making hydrogen bonds in the active site, as observed by the docking and molecular dynamics simulation studies. These results pave the path for further investigation of these molecules for their anti-leishmanial activities.

Structure of Orthoreovirus RNA Chaperone σNS, a Component of Viral Replication Factories.

The reovirus σNS RNA-binding protein is required for formation of intracellular compartments during viral infection that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of an R6A mutant of σNS that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure of selenomethionine-substituted σNS-R6A reveals that the mutant protein forms a stable antiparallel dimer, with each subunit having a well-folded central core and a projecting N-terminal arm. The dimers interact with each other by inserting the N-terminal arms into a hydrophobic pocket of the neighboring dimers on either side to form a helical assembly that resembles filaments of WT σNS in complex with RNA observed using cryo-EM. The interior of the crystallographic helical assembly is positively charged and of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same hydrophobic pocket as the N-terminal arm, as demonstrated in the crystal structure of σNS-R6A in complex with bile acid, suggesting that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS. This idea is supported by the structure of σNS lacking the N-terminal arm. We discovered that σNS displays RNA helix destabilizing and annealing activities, likely essential for presenting mRNA to the viral RNA-dependent RNA polymerase for genome replication. The RNA chaperone activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

Biochemical and structural basis for Moraxella catarrhalis enoyl-acyl carrier protein reductase (FabI) inhibition by triclosan and estradiol.

The enoyl-acyl carrier protein reductase (ENR) is an established drug target and catalyzes the last reduction step of the fatty acid elongation cycle. Here, we report the crystal structures of FabI from Moraxella catarrhalis (McFabI) in the apo form, binary complex with NAD+ and ternary complex with NAD + -triclosan (TCL) determined at 2.36, 2.12 and 2.22 Å resolutions, respectively. The comparative study of these three structures revealed three different conformational states for the substrate-binding loop (SBL), including an unstructured intermediate, a structured intermediate and a closed conformation in the apo, binary and ternary complex forms, respectively; indicating the flexibility of SBL during the ligand binding. Virtual screening has suggested that estradiol cypionate may be a potential inhibitor of McFabI. Subsequently, estradiol (EST), the natural form of estradiol cypionate, was assessed for its FabI-binding and -inhibition properties. In vitro studies demonstrated that TCL and EST bind to McFabI with high affinity (KD = 0.038 ± 0.004 and 5 ± 0.06 µM respectively) and inhibit its activity (Ki = 62.93 ± 3.95 nM and 25.97 ± 1.93 µM respectively) and suppress the growth of M. catarrhalis. These findings reveal that TCL and EST inhibit the McFabI activity and thereby affect cell growth. This study suggests that estradiol may be exploited as a novel scaffold for the designing and development of more potential FabI inhibitors.

MD simulation and MM/PBSA identifies phytochemicals as bifunctional inhibitors of SARS-CoV-2.

The global spread of SARS-CoV-2 has resulted in millions of fatalities worldwide, making it crucial to identify potent antiviral therapeutics to combat this virus. We employed structure-assisted virtual screening to identify phytochemicals that can target the two proteases which are essential for SARS-CoV-2 replication and transcription, the main protease and papain-like protease. Using virtual screening and molecular dynamics, we discovered new phytochemicals with inhibitory activity against the two proteases. Isoginkgetin, kaempferol-3-robinobioside, methyl amentoflavone, bianthraquinone, podocarpusflavone A, and albanin F were shown to have the best affinity and inhibitory potential among the compounds, and can be explored clinically for use as inhibitors of novel coronavirus SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Alphavirus antivirals targeting RNA-dependent RNA polymerase domain of nsP4 divulged using surface plasmon resonance.

Alphaviruses are continuously re-emerging and pose a global threat to human health and currently no antiviral drug is commercially available for alphaviral infections. Alphavirus non-structural protein nsP4, which possesses RNA-dependent RNA polymerase (RdRp) activity, is a potential antiviral target. To date, no antiviral drug is commercially available against alphaviruses. Since RdRp is the key virus-specific enzyme involved in viral genome replication, this study identifies and validates the antiviral efficacy of small molecules targeting alphavirus RdRp. Purified nsP4 was characterized using the surface plasmon resonance (SPR) assay, and the binding affinities of divalent metal ions, ribonucleotides, and in vitro transcribed viral RNA oligonucleotides were obtained in the micromolar (µm) range. Further, four potential inhibitors, piperine (PIP), 2-thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA), were identified against nsP4 RdRp using a molecular docking approach. The SPR assay validated the binding of PIP, 2TU, PZA, and CGA to purified nsP4 RdRp with KD of 0.08, 0.13, 0.66, and 9.87 µm, respectively. Initial testing of these molecules as alphavirus replication inhibitors was done using SINV-IRES-Luc virus. Detailed assessment of antiviral efficacy of molecules against CHIKV was performed by plaque reduction assay, qRT-PCR, and immunofluorescence assay. PIP, 2TU, PZA, and CGA showed antiviral potency against CHIKV with EC50 values of 6.68, 27.88, 36.26, and 53.62 µm, respectively. This study paves the way towards the development of novel broad-spectrum alphavirus antivirals targeting nsP4 RdRp.

Chikungunya virus titration, detection and diagnosis using N-Acetylglucosamine (GlcNAc) specific lectin based virus capture assay.

Re-emergence and global expansion of Chikungunya virus (CHIKV) from Africa to Indian Subcontinent in 2013, has significantly resulted in chronic morbidities in infected individuals. The burden of CHIKV on human population is still uncertain, owing to lack of vaccine and underdiagnosis. Due to the absence of vaccine or antiviral therapeutics, timely diagnosis and detection of CHIKV is vital for minimizing virus transmission. Commercially available diagnostic and titration kits relies on the traditional methods such as real-time PCR (RT-PCR), serodiagnostic assays, and plaque assay, which are expensive, time-consuming and technically challenging. To overcome these limitations and to increase the diagnostic coverage of CHIKV infections, a rapid and economical antigen capture assay has been developed in this study for serological diagnosis of CHIKV, using tamarind chitinase (chi)-like lectin (TCLL). TCLL extracted and purified from tamarind seeds (Tamarindus indica), has been reported recently to bind to N-acetylglucosamine (GlcNAc) containing glycan on the envelope protein of virus. Evaluation of antigen capture assay for serological diagnosis of CHIKV signified that the developed assay is able to detect CHIKV in both laboratory and clinical samples efficiently. Furthermore, a standard graph using different concentrations of CHIKV has been established using samples with known virus titer, to assist in quantification of viral load in a given sample. The feasibility of antigen capture assay for broad-spectrum diagnosis of alphaviral infections was evaluated using Sindbis virus (SINV) belonging to the same alphavirus genus, and the results obtained were in agreement with those of CHIKV. In summary, the developed glycan-based virus capture assay can be potentially applied as point-of-care routine diagnostic and titration assay for CHIKV as well for other re-emerging alphaviral infections.

Sleep Promotion among Critically Ill Patients: Earplugs/Eye Mask versus Ocean Sound-A Randomized Controlled Trial Study.

BACKGROUND: Poor sleep quality is common in the intensive care unit (ICU), where several factors including environmental factors contribute to sleep deprivation. OBJECTIVE: This study aims to assess and compare the effectiveness of earplugs and eye mask versus ocean sound on sleep quality among ICU patients. DESIGN: A true experimental crossover design was used. Setting. Medical ICU of the Maharishi Markandeshwar Institute of Medical Sciences and Research Hospital, Mullana, India. Participants. Sixty-eight patients admitted in the medical ICU were randomly allocated by lottery methods into group 1 and group 2. METHODS: Nocturnal nine-hour (10 : 00 pm to 7 : 00 am) for a four-night period were measured. Earplugs and eye mask versus ocean sound were crossed over between two groups. Subjective sleep quality of four nights was assessed using a structured sleep quality scale. Scores for each question range from 0 to 3, with a higher score indicating poor sleep quality. RESULTS: Repeated measures ANOVA showed that there was a significant change in the sleep quality score (p=0.001), which showed that sleep quality score was improved after the administration of earplugs and eye mask and ocean sound. Fisher's LSD post hoc comparison showed a significant difference (p=0.001). CONCLUSION: Earplugs and eye mask were better than ocean sound in improving sleep quality. Earplugs, eye mask, and ocean sound are safe and cost effective, which could be used as an adjuvant to pharmacological interventions to improve sleep quality among ICU patients. However, further research in this area needs to be conducted. This trial is registered with NCT03215212.

Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis.

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43 kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein-ligand complex structures were subjected to 50 ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.