An Immunoinformatics-Based Study of Mycobacterium tuberculosis Region of Difference-2 Uncharacterized Protein (Rv1987) as a Potential Subunit Vaccine Candidate for Preliminary Ex Vivo Analysis.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein's capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1ß, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.

Metal regulation of Mycobacterium tuberculosis SufB intein splicing at the host-pathogen crossroad.

Intein sequences self-excise from precursor proteins to generate functional proteins in various organisms. Thus, regulation of intein splicing at the host-pathogen interface can determine the fate of infection by controlling generation of essential proteins in microbes. For instance, Mycobacterium tuberculosis (Mtu) SufB intein splicing is crucial for the functionality of SUF complex. This multiprotein system is the sole pathway for [Fe-S] cluster biogenesis in mycobacteria during oxidative stress and Fe starvation. Although metal toxicity and metal starvation are components of host immunity, correlation of metal stress to Mtu SufB intein splicing is missing till date. Current study examines the splicing and N-terminal cleavage reactions of Mtu SufB precursor protein in presence of micronutrient metal ions like Zn+2, Cu+2, and Fe+3/+2. A known intein splicing inhibitor Pt+4 was also tested to support its proposed role as an anti-TB agent. Mtu SufB precursor protein exhibited significant attenuation of splicing and N-terminal cleavage reactions across different concentration ranges for Pt+4, Cu+2, Zn+2, while Fe+3 interaction resulted in precursor accumulation. UV-Vis spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), Tryptophan fluorescence assay, and dynamic light scattering (DLS) techniques analyzed metal-protein interaction. Mutagenesis experiments and Ellman's assay identified plausible metal co-ordination sites within Mtu SufB protein. Analyzing the metal effect on Mtu SufB splicing may provide elemental information about the fate of mycobacterial infection, and a probable mechanism to attenuate intracellular survival of Mtu. Current research hints at the host regulatory mechanism on SufB splicing in its native environment and a likely target for developing next-generation anti-TB drugs.

In silico and in vitro study of Mycobacterium tuberculosis H37Rv uncharacterized protein (RipD): an insight on tuberculosis therapeutics.

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is responsible for the highest global health problem, with the deaths of millions of people. With prevalence of multiple drug resistance (MDR) strains and extended therapeutic times, it is important to discover small molecule inhibitors against novel hypothetical proteins of the pathogen. In this study, a virtual screening protocol was carried out against MtbH37Rv hypothetical protein RipD (Rv1566c) for the identification of potential small molecule inhibitors. The 3D model of the protein structure binding site was used for virtual screening (VS) of inhibitors from the Pathogen Box, followed by its validation through a molecular docking study. The stability of the protein-ligand complex was assessed using a 150 ns molecular dynamics simulation. MM-PBSA and MM-GBSA are the two approaches that were used to perform the trajectory analysis and determine the binding free energies, respectively. The ligand binding was observed to be stable across the entire time frame with an approximate binding free energy of -22.9916 kcal/mol. The drug-likeness of the inhibitors along with a potential anti-tuberculosis compound was validated by ADMET prediction software. Furthermore, a CFU inhibition assay was used to validate the best hit compound's in vitro inhibitory efficacy against a non-pathogenic Mycobacterium smegmatis MC2155 under low nutrient culture conditions. The study reported that the compound proposed in our study (Pathogen Box ID: MMV687700) will be useful for the identification of potential inhibitors against Mtb in future.

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines.

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

Epidemiological Analysis of SARS-CoV-2 Transmission Dynamics in the State of Odisha, India: A Yearlong Exploratory Data Analysis.

COVID-19 remains a matter of global public health concern. Previous research suggested the association between local environmental factors and viral transmission. We present a multivariate observational analysis of SARS-CoV-2 transmission in the state of Odisha, India, hinting at a seasonal activity. We aim to investigate the demographic characteristics of COVID-19 in the Indian state of Odisha for two specific timelines in 2020 and 2021. For a comparative outlook, we chose similar datasets from the state of New York, USA. Further, we present a critical analysis pertaining to the effects of environmental factors and the emergence of variants on SARS-CoV-2 transmission and persistence. We assessed the datasets for confirmed cases, death, age, and gender for 29 February 2020 to 31 May 2020, and 1 March 2021 to 31 May 2021. We determined the case fatalities, crude death rates, sex ratio, and incidence rates for both states along with monthly average temperature analysis. A yearlong epi-curve analysis was conducted to depict the coronavirus infection spread pattern in the respective states. The Indian state of Odisha reported a massive 436,455 confirmed cases and 875 deaths during the 2021 timeline as compared to a mere 2223 cases and 7 deaths during the 2020 timeline. We further discuss the demographic and temperature association of SARS-CoV-2 transmission during early 2020 and additionally comment on the variant-associated massive rise in cases during 2021. Along with the rapid rise of variants, the high population density and population behavior seem to be leading causes for the 2021 pandemic, whereas factors such as age group, gender, and average local temperature were prominent during the 2020 spread. A seasonal occurrence of SARS-CoV-2 transmission is also observed from the yearlong epidemiological plot. The recent second wave of COVID-19 is a lesson that emphasizes the significance of continuous epidemiological surveillance to predict the relative risk of viral transmission for a specific region.

Metal effect on intein splicing: A review.

Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from the precursor protein, ligating concomitant extein residues in a process called protein splicing. Post-translational auto-removal of inteins remain critical for the generation of active proteins. The perspective of inteins in science is a robust field of research, however fundamental studies centralized upon splicing regulatory mechanism are imperative for addressing more intricate issues. Controlled engineering of intein splicing has many applications; intein inhibition can facilitate novel drug design, while activation of intein splicing is exploited in protein purification. This paper provides a comprehensive review of the past and recent advances in the splicing regulation via metal-intein interaction. We compare the behavior of different metal ions on diverse intein systems. Though metals such as Zn, Cu, Pt, Cd, Co, Ni exhibit intein inhibitory effect heterogeneously on different inteins, divalent metal ions such as Ca and Mg fail to do so. The observed diversity in the metal-intein interaction arises mostly due to intein polymorphism and variations in atomic structure of metals. A mechanistic understanding of intein regulation by metals in native as well as synthetically engineered intein systems may yield potent intein inhibitors via direct or indirect approach.

Computational discovery and ex-vivo validation study of novel antigenic vaccine candidates against tuberculosis.

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.

Gold Nanoparticles Augment N-Terminal Cleavage and Splicing Reactions in Mycobacterium tuberculosis SufB.

Protein splicing is a self-catalyzed event where the intervening sequence intein cleaves off, joining the flanking exteins together to generate a functional protein. Attempts have been made to regulate the splicing rate through variations in temperature, pH, and metals. Although metal-regulated protein splicing has been more captivating to researchers, metals were shown to only inhibit splicing reactions that confine their application. This is the first study to show the effect of nanoparticles (NPs) on protein splicing. We found that gold nanoparticles (AuNPs) of various sizes can increase the splicing efficiency by more than 50% and the N-terminal cleavage efficiency by more than 45% in Mycobacterium tuberculosis SufB precursor protein. This study provides an effective strategy for engineering splicing-enhanced intein platforms. UV-vis absorption spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy (TEM) confirmed AuNP interaction with the native protein. Quantum mechanics/molecular mechanics (QM/MM) analysis suggested a significant reduction in the energy barrier at the N-terminal cleavage site in the presence of gold atom, strengthening our experimental evidence on heightened the N-terminal cleavage reaction. The encouraging observation of enhanced N-terminal cleavage and splicing reaction can have potential implementations from developing a rapid drug delivery system to designing a contemporary protein purification system.

Inteins in Science: Evolution to Application.

Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

Fisetin prevents the aging-associated decline in relative spectral power of α, ß and linked MUA in the cortex and behavioral alterations.

Mental health in old age is of great concern due to the increased incidence of neurological and psychiatric diseases. With age, probability of cognitive and behavioral deficits increase and the prognosis deteriorates. Although a few in vitro studies have reported that flavonoid fisetin is beneficial for healthy aging, its effect on deteriorating mental health with aging in vivo is very limited and poorly understood. The brain aging is physiologically characterized by electroencephalograph (EEG) wave frequency, power, and distribution. Brain oscillatory waves from neural tissue get altered by various sensory-cognitive inputs. Besides, the fast-wave α(8-12 Hz)- and ß(12-28 Hz)-oscillations are associated with coordination and indeed deal with complex behavioral performances. Therefore, the effect of fisetin supplementation on age-associated EEG mean cortical spectral power in α- and ß-oscillations, multi-unit activity (MUA) count were studied in vivo which was not addressed so far. Besides, age-associated cognitive and behavioral alterations were also studied. The relative spectral power of α and ß declined along with the MUA count in aged rats compared to young. However, supplementing fisetin for four weeks has improved relative α-power, ß-power, and MUA count in aged rats. Also, fisetin supplemented aged rats showed significantly improved cognitive and behavioral performances than aged controls. These findings demonstrated the relative cortical spectral power in α-, ß-oscillations, and MUA count change in aged rats and that some of these changes and behavioral alterations may be partly as a result of oxidative stress, which was prevented significantly in fisetin supplemented aged rats. Thus, fisetin boosted mental health in the aged animals.

Electron Transfer Directed Antibacterial Properties of Graphene Oxide on Metals.

Nanomaterials such as silver nanoparticles and graphene-based composites are known to exhibit biocidal activities. However, interactions with surrounding medium or supporting substrates can significantly influence this activity. Here, it is shown that superior antimicrobial properties of natural shellac-derived graphene oxide (GO) coatings is obtained on metallic films, such as Zn, Ni, Sn, and steel. It is also found that such activities are directly correlated to the electrical conductivity of the GO-metal systems; the higher the conductivity the better is the antibacterial activity. GO-metal substrate interactions serve as an efficient electron sink for the bacterial respiratory pathway, where electrons modify oxygen containing functional groups on GO surfaces to generate reactive oxygen species (ROS). A concerted effect of nonoxidative electron transfer mechanism and consequent ROS mediated oxidative stress to the bacteria result in an enhanced antimicrobial action of naturally derived GO-metal films. The lack of germicidal effect in exposed cells for GO supported on electrically nonconductive substrates such as glass corroborates the above hypothesis. The results can lead to new GO coated antibacterial metal surfaces important for environmental and biomedical applications.

Chemical Vapor Deposited Few-Layer Graphene as an Electron Field Emitter.

Chemical vapor deposition (CVD) growth of graphene on polycrystalline copper (Cu) foil in a low pressure conditions has been presented, aiming to achieve the highest quality with large-scale fabrications, which requires comprehensive understanding and effective controlling of the growth process. Herein, few-layer graphene (FLG) films with large-domain sizes were grown on Cu metal catalyst substrates using a vertical mass-flow hot-filament CVD reactor, with the intention of large scale production, by optimizing the CVD system and three of the process parameters: (i) gas flow compositions, (ii) substrate annealing time and (iii) graphene deposition time. The detailed scanning electron microscope and Raman spectroscopy analysis indicate that all the above mentioned process parameters affect growth of FLG film on Cu substrate. The presence of two intense peaks, G and 2D-band at 1583.6 and 2702.6 cm⁻¹ for synthesized sample at optimized conditions (H2/CH4 ratio of 50:1 at graphene deposition time of 10 minutes and substrate annealed time for 20 minutes) revealed the formation of FLG films with large domain size. These graphene films on Cu have shown the room temperature field electron emission characteristics, hence appears to be prospective candidate for vacuum nanoelectronics.

Electrical Characteristics of Horizontally and Vertically Oriented Few-Layer Graphene on Si-Based Dielectrics.

Horizontally and vertically oriented few-layer graphenes have been synthesized directly on SiO2 coated Si substrates via thermal and hot-filament chemical vapor deposition, respectively. The effect of the direction of mass flow on the fabrication of graphene film is analysed and a plausible mechanism is proposed. The graphene/p-Si heterojunction is fabricated and tested for its potential in optoelectronic devices. Rectification behaviour is observed at the interface of graphene/p-Si under dark conditions. A dark current of 1.1 mA with an ideality factor of 1.5, in addition of a high rectification ratio of 15.99 at ± 0.5 V is found for the vertically oriented graphene/p-Si heterojunction.

Optimizing Performance Parameters of Chemically-Derived Graphene/p-Si Heterojunction Solar Cell.

Chemically-derived graphene have been synthesized by modified Hummers method and reduced using sodium borohydride. To explore the potential for photovoltaic applications, graphene/p-silicon (Si) heterojunction devices were fabricated using a simple and cost effective technique called spin coating. The SEM analysis shows the formation of graphene oxide (GO) flakes which become smooth after reduction. The absence of oxygen containing functional groups, as observed in FT-IR spectra, reveals the reduction of GO, i.e., reduced graphene oxide (rGO). It was further confirmed by Raman analysis, which shows slight reduction in G-band intensity with respect to D-band. Hall effect measurement confirmed n-type nature of rGO. Therefore, an effort has been made to simu- late rGO/p-Si heterojunction device by using the one-dimensional solar cell capacitance software, considering the experimentally derived parameters. The detail analysis of the effects of Si thickness, graphene thickness and temperature on the performance of the device has been presented.

SufB intein of Mycobacterium tuberculosis as a sensor for oxidative and nitrosative stresses.

Inteins are mobile genetic elements that self-splice at the protein level. Mycobacteria have inteins inserted into several important genes, including those corresponding to the iron-sulfur cluster assembly protein SufB. Curiously, the SufB inteins are found primarily in mycobacterial species that are potential human pathogens. Here we discovered an exceptional sensitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses when expressed in Escherichia coli. This effect results from predisposition of the intein's catalytic cysteine residues to oxidative and nitrosative modifications. Experiments with a fluorescent reporter system revealed that reactive oxygen species and reactive nitrogen species inhibit SufB extein ligation by forcing either precursor accumulation or N-terminal cleavage. We propose that splicing inhibition is an immediate, posttranslational regulatory response that can be either reversible, by inducing precursor accumulation, or irreversible, by inducing N-terminal cleavage, which may potentially channel mycobacteria into dormancy under extreme oxidative and nitrosative stresses.

Transparent conductive multiwall carbon nanotubes-polymer composite for electrode applications.

Disperse Multiwall carbon nanotubes (MWCNTs) are incorporated aqueous N-hydroxy methyl acrylamide, which is subjected to crosslinking to develop a transparent conductive composite free standing film. The effects of the concentration of MWCNTs and temperature on optical and electrical properties of nano-composites are investigated. Interestingly, only 0.06 mg/ml of MWCNTs is sufficient to reach the percolation threshold (Phi) for transition in electrical conductivity up to 10(-4) S/cm, with a visible transmittance over 85%, which is well above the reported for such a low level of MWCNTs loading. The electrical conductivity of the composite was measured at 120 degrees C. It has been observed that electrical conductivity increases significantly with the increase in temperature, signifying the semiconducting nature of nano-composites. Finally, current-voltage (I-V) characteristics show liner behaviour, confirms Ohmic nature of nano-composites and metal contact.

Fabrication of bi-layer graphene and theoretical simulation for its possible application in thin film solar cell.

High quality graphene film is fabricated using mechanical exfoliation of highly-oriented pyrolytic graphite. The graphene films on glass substrates are characterized using field-emission scanning electron microscopy, atomic force microscopy, Raman spectroscopy, UV-vis spectroscopy and Fourier transform infrared spectroscopy. A very high intensity ratio of 2D to G-band (to approximately 1.67) and narrow 2D-band full-width at half maximum (to approximately 40 cm(-1)) correspond to the bi-layer graphene formation. The bi-layer graphene/p-GaN/n-InGaN/n-GaN/GaN/sAl2O3 system is studied theoretically using TCAD Silvaco software, in which the properties of exfoliated bi-layer graphene are used as transparent and conductive film, and the device exhibits an efficiency of 15.24% compared to 13.63% for ITO/p-GaN/n-InGaN/n-GaN/GaN/Al2O3 system.

Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein.

Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called "leave-one-outs" using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents.