Characterization of a putative ribosome binding site at the 5' untranslated region of bovine heat shock protein 90.

Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. Internal ribosome entry site (IRES) mediated alternative translation initiations are emerging as unique mechanisms. Present study is aimed to indentify a functional short 92 base pair length putative sequence located at the 5' untranslated region of bovine heat shock protein 90 AA1 (Hsp90AA1) may interact with ribosomal as well as eukaryotic initiation factor binding site. Here we have predicted both the two and three dimensional structures of bovine Hsp90AA1 IRES (MF400854) element with their respective free energy. Molecular interactions between bovine RPS5 and IRES have been determined after the preparation of docking complex of IRES bound RPS5. Structure of bovine ribosomal translational initiation factor (TIF) has also been determined and docked with IRES. Molecular interaction between bovine TIF and IRES was analyzed from the complex structure. We further detected the relative expression efficiency of the viral (original) in relation with Hsp90AA1 IRES-driven GFP expression, which revealed that efficiency under the control of identified bovine Hsp90AA1 IRES was slightly lower than viral origin. It was also noted that identified bovine HSP90 IRES may increase the expression level of GFP under in vitro heat stressed condition.

Characterization of a Class A ß-Lactamase from Francisella tularensis (Ftu-1) Belonging to a Unique Subclass toward Understanding AMR.

ß-lactamase production with vast catalytic divergence in the pathogenic strain limits the antibiotic spectrum in the clinical environment. Class A carbapenemase shares significant sequence similarities, structural features, and common catalytic mechanisms although their resistance spectrum differs from class A ß-lactamase in carbapenem and monobactam hydrolysis. In other words, it limited the antibiotic treatment option against infection, causing carbapenemase-producing superbugs. Ftu-1 is a class A ß-lactamase expressed by the Francisella tularensis strain, a potent causative organism of tularemia. The chromosomally encoded class A ß-lactamase shares two conserved cysteine residues, a common characteristic of a carbapenemase, and a distinctive class in the phylogenetic tree. Complete biochemical and biophysical characterization of the enzyme was performed to understand the overall stability and environmental requirements to perform optimally. To comprehend the enzyme-drug interaction and its profile toward various chemistries of ß-lactam and ß-lactamase inhibitors, comprehensive kinetic and thermodynamic analyses were conducted using various ß-lactam drugs. The dynamic property of Ftu-1 ß-lactamase was also predicted using molecular dynamics (MD) simulation to compare its loop flexibility and ligand binding with other related class A ß-lactamases. Overall, this study fosters a comprehensive understanding of Ftu-1, proposed to be an intermediate class by characterizing its kinetic profiling, stability by biochemical and biophysical methodologies, and susceptibility profiling. This understanding would be beneficial for the design of new-generation therapeutics.

Differential Binding of Carbapenems with the AdeABC Efflux Pump and Modulation of the Expression of AdeB Linked to Novel Mutations within Two-Component System AdeRS in Carbapenem-Resistant Acinetobacter baumannii.

Resistance-nodulation-division-type efflux system AdeABC plays an important role in carbapenem resistance among Acinetobacter baumannii. However, a knowledge gap is observed regarding the role of its regulator AdeRS in carbapenem-resistant A. baumannii (CRAB). This study effectively combines microbiological analysis with an in-silico structural approach to understand the contribution of AdeRS among CRAB (n = 38). Additionally, molecular docking was performed for the first time to study the interaction of FDA-approved carbapenems and pump inhibitor PAßN with the open and closed structure of AdeB at the three binding sites (periplasmic, proximal, distal). It was observed that open conformation of AdeB facilitates the binding of carbapenems and PAßN at entrance and proximal sites compared to the closed conformation. PAßN was found to block carbapenem interacting residues in AdeB, establishing its role as a competitive inhibitor of AdeB substrates. Overexpression of AdeABC was detected by q-RT-PCR among 29% of CRABs, and several mutations within AdeS (GLY186VAL, SER188PHE, GLU121LYS, VAL255ILE) and AdeR (VAL120ILE, ALA136VAL) were detected by sequencing. The sequence and structure-based study of AdeRS was performed to analyze the probable effect of these mutations on regulation of the two-component system (TCS), especially, utilizing its three-dimensional structure. AdeS mutations inhibited the transfer of a phosphate group to AdeR, preventing the binding of AdeR to the intercistronic region, leading to overexpression of AdeABC. The elucidation of the role of mutations in AdeRS improves our understanding of TCS-based regulation. Identification of the key residues of AdeB interacting with carbapenems and PAßN may help in future designing of novel inhibitors. IMPORTANCE AdeABC is an important efflux pump in A. baumannii that plays a role in resistance toward different antibiotics including the "last resort" antibiotic, carbapenem. This pump is regulated by a two-component system, AdeRS. To understand the binding of carbapenems with AdeABC and pump inhibition by PAßN, we analyzed for the first time the possible atomic level interactions of carbapenems and PAßN with AdeB. In the current study, AdeRS-associated novel mutations in clinical A. baumannii are reported for the first time, and a sequence-structure based in-silico approach was used to interpret their role in AdeABC overexpression, leading to carbapenem resistance. None of the previous studies had undertaken both these aspects simultaneously. This study analyzes the open and closed conformation of AdeB, their binding with carbapenems, and key residues involved in it. This helps in visualizing the plausible atomic level causes of pump inhibition driving the discovery of novel inhibitors.

Synthesis of Dihydrobenzofuro[3,2-b]chromenes as Potential 3CLpro Inhibitors of SARS-CoV-2: A Molecular Docking and Molecular Dynamics Study.

The recent emergence of pandemic of coronavirus (COVID-19) caused by SARS-CoV-2 has raised significant global health concerns. More importantly, there is no specific therapeutics currently available to combat against this deadly infection. The enzyme 3-chymotrypsin-like cysteine protease (3CLpro) is known to be essential for viral life cycle as it controls the coronavirus replication. 3CLpro could be a potential drug target as established before in the case of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). In the current study, we wanted to explore the potential of fused flavonoids as 3CLpro inhibitors. Fused flavonoids (5a,10a-dihydro-11H-benzofuro[3,2-b]chromene) are unexplored for their potential bioactivities due to their low natural occurrences. Their synthetic congeners are also rare due to unavailability of general synthetic methodology. Here we designed a simple strategy to synthesize 5a,10a-dihydro-11H-benzofuro[3,2-b]chromene skeleton and it's four novel derivatives. Our structural bioinformatics study clearly shows excellent potential of the synthesized compounds in comparison to experimentally validated inhibitor N3. Moreover, in-silico ADMET study displays excellent druggability and extremely low level of toxicity of the synthesized molecules. Further, for better understanding, the molecular dynamic approach was implemented to study the change in dynamicity after the compounds bind to the protein. A detailed investigation through clustering analysis and distance calculation gave us sound comprehensive data about their molecular interaction. In summary, we anticipate that the currently synthesized molecules could not only be a potential set of inhibitors against 3CLpro but also the insights acquired from the current study would be instrumental in further developing novel natural flavonoid based anti-COVID therapeutic spectrums.

In silico modeling revealed new insights into the mechanism of action of enzyme 2'-5'-oligoadenylate synthetase in cattle.

The innate immune system has an important role in developing the initial resistance to virus infection, and the ability of oligoadenylate synthetase to overcome viral evasion and enhance innate immunity is already established in humans. In the present study, we have tried to explore the molecular and structural variations present in Sahiwal (indigenous) and crossbred (Frieswal) cattle to identify the molecular mechanism of action of OAS1 gene in activation of innate immune response. The significant changes in structural alignment in terms of orientation of loops, shortening of ß-sheets and formation of 3-10 α-helix was noticed in Sahiwal and Frieswal cattle. Further, it has been observed that OAS1 from Sahiwal had better binding with APC and DTP ligand than Frieswal OAS1. A remarkable change was seen in orientation at the nucleoside base region of both the ligands, which are bound with OAS1 protein from Frieswal and Sahiwal cattle. The Molecular Dynamic study of apo and ligand complex structures was provided more insight towards the stability of OAS1 from both cattle. This analysis displayed that the Sahiwal cattle protein has more steady nature throughout the simulation and has better binding towards Frieswal in terms of APC and DTP binding. Thus, OAS1 protein is the potential target for explaining the innate immune response in Sahiwal than Frieswal.Communicated by Ramaswamy H. Sarma.

Understanding structure-based dynamic interactions of antihypertensive peptides extracted from food sources.

Functional foods are emerging as essential healthy nutritional component due to their abundant wellbeing benefits. Especially the food-derived peptides are considered as key components for playing their biologically active roles. One such robust therapeutics that already exploited with food peptides that help treating high blood pressure via targeting Angiotensin-Converting Enzyme (ACE). This in silico study demonstrated the inhibitory potential of antihypertensive peptides derived from food sources. This study involves an intensive structure-based analysis of enzyme-peptide interactions using Molecular Dynamics (MD) simulations. Interestingly, this study will help us to get deeper understanding on how food peptides achieve successful inhibition of ACE. In this study, the peptide-enzyme complexes revealed two binding pockets, A and B, on either side of the active site Zn atom. Pocket B has a smaller binding site volume than pocket A, comprised of ß-sheets and the active site opening cleft. The interface of the binding sites showed that the enzyme structure was negative to neutral charge, and the peptide structure was positive to neutral charge. The dynamics of complex structures of seven highly potential peptides were performed for 20 ns each at 300 K. Comparative analysis of RMSD, RMSF and binding energies show the enzyme-peptide complexes and the overall stability of apo-enzyme. Importantly, two peptides AFKAWAVAR and IWHHTF showed the highest variation in their RMSD as compared to the apo-enzyme. This study will further be useful for the assessment of the characteristics to predict novel inhibitory peptides that can be generated from food proteins.Communicated by Ramaswamy H. Sarma.

Anti-hypertensive Peptide Predictor: A Machine Learning-Empowered Web Server for Prediction of Food-Derived Peptides with Potential Angiotensin-Converting Enzyme-I Inhibitory Activity.

Angiotensin converting enzyme-I (ACE-I) is a key therapeutic target of the renin-angiotensin-aldosterone system (RAAS), the central pathway of blood pressure regulation. Food-derived peptides with ACE-I inhibitory activities are receiving significant research attention. However, identification of ACE-I inhibitory peptides from different food proteins is a labor-intensive, lengthy, and expensive process. For successful identification of potential ACE-I inhibitory peptides from food sources, a machine learning and structural bioinformatics-based web server has been developed and reported in this study. The web server can take input in the FASTA format or through UniProt ID to perform the in silico gastrointestinal digestion and then screen the resulting peptides for ACE-I inhibitory activity. This unique platform provides elaborated structural and functional features of the active peptides and their interaction with ACE-I. Thus, it can potentially enhance the efficacy and reduce the time and cost in identifying and characterizing novel ACE-I inhibitory peptides from food proteins. URL: http://hazralab.iitr.ac.in/ahpp/index.php.

Understanding the molecular interactions of inhibitors against Bla1 beta-lactamase towards unraveling the mechanism of antimicrobial resistance.

This study evaluated the inhibitory potential of various beta-lactamase inhibitors such as mechanism-based inhibitors (MBIs), carbapenems, monobactam, and non-beta-lactam inhibitors against Bla1, a class-A beta-lactamase encoded by Bacillus anthracis. The binding potential of different inhibitors was estimated using competitive kinetic assay, isothermal titration calorimetry, and Biolayer interferometry. We observed that tazobactam has better inhibition among other MBIs with a characteristics inhibition dissociation constant of 0.51 ± 0.13 µM. Avibactam was also identified as good inhibitor with an inhibition efficiency of 0.6 ± 0.04 µM. All the MBIs (KD = 1.90E-04 M, 2.05E-05 M, 3.55E-04 M for clavulanate, sulbactam and tazobactam) showed significantly better binding potential than carbapenems (KD = 1.02E-03 M, 2.74E-03 M, 1.24E-03 M for ertapenem, imipenem and biapenem respectively). Molecular dynamics simulations were carried out using Bla1-inhibitor complexes to understand the dynamics and stability. The minimum inhibitory concentration (MIC) was carried out by taking various substrates and inhibitors, and later it was followed by cell viability assay. Together, our study helps develop a proper understanding of Bla1 beta-lactamase and its interaction with inhibitory molecules. This study would facilitate comprehending the catalytic divergence of beta-lactamases and the newly emergent resistant strains, focusing on the new generation of therapeutics being less prone to antimicrobial resistance.

Variations in the SDN Loop of Class A Beta-Lactamases: A Study of the Molecular Mechanism of BlaC (Mycobacterium tuberculosis) to Alter the Stability and Catalytic Activity Towards Antibiotic Resistance of MBIs.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for an immediate search for novel treatment strategies. Recently, BlaC, the principal beta-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. BlaC belongs to Ambler class A, which is generally susceptible to the beta-lactamase inhibitors currently used in clinics: tazobactam, sulbactam, and clavulanate. Alterations at Ser130 in conserved SDN loop confer resistance to mechanism-based inhibitors (MBIs) commonly observed in various clinical isolates. The absence of clinical evidence of S130G conversion in M. tuberculosis draws our attention to build laboratory mutants of S130G and S130A of BlaC. The study involving steady state, inhibition kinetics, and fluorescence microscopy shows the emergence of resistance against MBIs to the mutants expressing S130G and S130A. To understand the molecular reasoning behind the unavailability of such mutation in real life, we have used circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), molecular dynamics (MD) simulation, and stability-based enzyme activity to compare the stability and dynamic behaviors of native and S130G/A mutant form of BlaC. A significant decrease in melting temperature (BlaC T M 60°C, S130A T M 50°C, and S130G T M 45°C), kinetic instability at higher temperature, and comparative dynamic instability correlate the fact that resistance to beta-lactam/beta-lactamase inhibitor combinations will likely not arise from the structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be potentially applied as a part of a successful treatment regimen against M. tuberculosis.

A comprehensive characterization of novel CYP-BM3 homolog (CYP-BA) from Bacillus aryabhattai.

Functionalizing C-H bond poses one of the most significant challenges for chemists providing them with very few substrate-specific synthetic routes. Despite being incredibly plastic in their enzymatic ability, they are confined with deficient enzymatic action and limited explicitness of the substrates. In this study, we have endeavored to characterize novel cytochrome P450 from Bacillus aryabhattai (CYP-BA), a homolog of CYP P450-BM3, by taking interdisciplinary approaches. We conducted structure and sequence comparison to understand the conservation pattern for active site residues, conserved fold, evolutionary relationships among others. Molecular dynamics simulations were performed to understand the dynamic nature and interaction with the substrates. CYP-BA was successfully cloned, purified, and characterized. The enzyme's stability toward various physicochemical parameters was evaluated by UV-vis spectroscopy and Circular Dichroism (CD) spectroscopy. Various saturated fatty acids being the natural cytochrome P450 substrates were evaluated as catalytic efficiency of substrate oxidation by CYP-BA. The binding affinity of these natural substrates was monitored against CYP-BA by isothermal titration calorimetry (ITC). The catalytic performance of CYP-BA was satisfactory enough to proceed to the next step, that is, engineering to expand the substrate range to include polycyclic aromatic hydrocarbons (PAH). This is the first evidence of cloning, purifying and characterizing a novel homolog of CYP-BM3 to enable a better understanding of this novel biocatalyst and to provide a platform toward expanding its catalytic process through enzyme engineering.

An insight into the complete biophysical and biochemical characterization of novel class A beta-lactamase (Bla1) from Bacillus anthracis.

Bacillus anthracis, a potent pathogen of anthrax is becoming resistant to many beta-lactam antibiotics because of the expression of two chromosomally encoded beta-lactamases Bla1 and Bla2. Bla1 is a class A beta-lactamase whereas Bla2 is a Metallo beta-lactamase. In the current study, we have attempted in-detailed characterization of Bla1 beta-lactamase by taking interdisciplinary approaches. Our study includes structure and sequence comparison of this enzyme with other members of the class, to know the conservation pattern that includes active site residues, secondary structure, conserved fold, evolutionary relationships, etc. Dynamic characterizations of the enzyme, unfolding kinetics were determined with the help of Molecular dynamics simulation. Detailed enzyme stability and catalytic activity towards various physical (Temperature and pH), and chemical parameters (Urea, GnHCl) were performed. Together, our study helps to develop a proper understanding of this beta-lactamase by characterizing its biochemical, biophysical, dynamic, kinetic and thermodynamic properties. This would help contribute towards a better understanding of beta-lactamase based AMR emergence.

Xylitol Production from Lignocellulosic Pentosans: A Rational Strain Engineering Approach toward a Multiproduct Biorefinery.

Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.

Database on spermatozoa transcriptogram of catagorised Frieswal crossbred (Holstein Friesian X Sahiwal) bulls.

Bull spermatozoa contain different functional genes and many of them plays important roles in different stages of spermatogenesis, spermatozoa kinetics, fertilization as well as embryonic development. RNA deep sequencing is one of the preferred tools for absolute quantification of messenger RNA. The intention of the current study was to investigate the abundance of spermatozoal transcripts in categorized Frieswal (Holstein-Friesian X Sahiwal) crossbred bull semen through RNA deep sequencing. A total 1546561 and 1019308 numbers of reads were identified among good and poor quality bull spermatozoa based on their conception rate. Post mapping with Bos taurus reference genome identified 1,321,236 and 842,022 number of transcripts among good and poor quality RNA libraries, respectively. However, a total number of 3510 and 6759 functional transcripts were identified among good and poor quality bull spermatozoa, respectively. Most of the identified transcripts were related to spermatozoa functions, embryonic development and other functional aspects of fertilization. Wet laboratory validation of the top five selected transcripts (AKAP4, PRM1, ATP2B4, TRIM71 and SLC9B2) illustrated the significant (p < 0.01) level of expression in the good quality crossbred bull semen than the poor quality counterparts. The present study with comprehensive profiling of spermatozoal transcripts provides a useful non-invasive tool to understand the causes of as well as an effective way to predict male infertility in crossbred bulls.

Identification of differentially expressed microRNAs in Sahiwal (Bos indicus) breed of cattle during thermal stress.

microRNAs (miRNAs) are a class of small non-coding RNAs that play key roles in post transcriptional gene regulation that influence various fundamental cellular processes, including the cellular responses during environmental stresses. However, perusal of literatures revealed few reports on the differential expression of miRNA during thermal stress in Indian native (Bos indicus) cattle breeds. The present investigation aimed to identify differentially expressed miRNAs during thermal stress in Sahiwal (Bos indicus) dairy cattle breed of India, adapted with tropical climate over a long period of time. Stress responses of the animals were characterized by determining various physiological as well as biochemical parameters and differential expression profile of major heat shock protein genes. Ion Torrent deep sequencing and CLC-genomic analysis identified a set of differentially expressed miRNAs during summer and winter seasons. Most of the identified differentially expressed miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family. Real-time quantification-based analysis of selected miRNAs revealed that bta-mir-1248, bta-mir-2332, bta-mir-2478, and bta-mir-1839 were significantly (p < 0.01) over expressed while bta-mir-16a, bta-let-7b, bta-mir-142, and bta-mir-425 were significantly (p < 0.01) under expressed during summer in comparison to winter. The present study enlists differentially expressed miRNAs at different environmental temperatures in Sahiwal (Bos indicus) that may be importance for further understanding the role of miRNAs on thermo-regulatory mechanisms.