Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.     

2,4,6-Triphenylaniline nanoemulsion formulation,optimization, and its application in type 2 diabetes mellitus

The secondary metabolite 2,4,6-triphenylaniline (TPA) was isolated from an endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis, that exhibited satisfactory in vitro antidiabetic activity for type 2 diabetes mellitus (T2DM). The TPA was encapsulated using nanoemulsion (NE) to overcome the problem of stability and permeability to increase its therapeutic applications. Response surface methodology (RSM) was used for the optimization of the variables given, such as hydrodynamic diameter, surface charge, and polydispersity index (PDI). TPA was encapsulated using an optimized ratio of olive oil and tween 80 (2:1) significantly affected the response variables including particle size (124.8 nm), ζ potential (−46.0 mV), and PDI (0.396), and the encapsulation efficiency was found to be 95.93%. The TPA-loaded NE after MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis showed nontoxic effects on L929 normal cell lines (areolar and adipose subcutaneous connective tissue of Mus musculus) with a viable percentage of 92%. In vitro release study revealed the slow and sustained release of the TPA over 48 hrs from NE under the Fickian diffusion mechanism and followed the Higuchi model for release kinetics. Further, the percentage of α-glucosidase and α-amylase inhibition rate of TPA-loaded NE was found to be 78.5 and 43.42%, respectively. The present study, therefore, can aid in the development of a novel drug delivery system as a therapeutic approach to T2DM.     

Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.     

Filament organization of the bacterial actin MreB is dependent on the nucleotide state

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5^WT and an ATPase-deficient mutant, ScMreB5^E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5^E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.     

Oxidative Damage in Muscular Dystrophy Correlates with the Severity of the Pathology: Role of Glutathione Metabolism

Muscular dystrophies (MDs) such as Duchenne muscular dystrophy (DMD), sarcoglycanopathy (Sgpy) and dysferlinopathy (Dysfy) are recessive genetic neuromuscular diseases that display muscle degeneration. Although these MDs have comparable endpoints of muscle pathology, the onset, severity and the course of these diseases are diverse. Different mechanisms downstream of genetic mutations might underlie the disparity in these pathologies. We surmised that oxidative damage and altered antioxidant function might contribute to these differences. The oxidant and antioxidant markers in the muscle biopsies from patients with DMD (n = 15), Sgpy (n = 15) and Dysfy (n = 15) were compared to controls (n = 10). Protein oxidation and lipid peroxidation was evident in all MDs and correlated with the severity of pathology, with DMD, the most severe dystrophic condition showing maximum damage, followed by Sgpy and Dysfy. Oxidative damage in DMD and Sgpy was attributed to the depletion of glutathione (GSH) and lowered antioxidant activities while loss of GSH peroxidase and GSH-S-transferase activities was observed in Dysfy. Lower GSH level in DMD was due to lowered activity of gamma-glutamyl cysteine ligase, the rate limiting enzyme in GSH synthesis. Similar analysis in cardiotoxin (CTX) mouse model of MD showed that the dystrophic muscle pathology correlated with GSH depletion and lipid peroxidation. Depletion of GSH prior to CTX exposure in C2C12 myoblasts exacerbated oxidative damage and myotoxicity. We deduce that the pro and anti-oxidant mechanisms could be correlated to the severity of MD and might influence the dystrophic pathology to a different extent in various MDs. On a therapeutic note, this could help in evolving novel therapies that offer myoprotection in MD.     

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.     

Secondary metabolite as therapeutic agent from endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis

Endophytic fungi, especially from mangrove plants, are rich source of secondary metabolites, which plays a major role in various pharmacological actions preferably in cancer and bacterial infections. To perceive its role in antidiabetic activity we isolated and tested the metabolites derived from a novel strain Alternaria longipes strain VITN14G obtained from mangrove plant Avicennia officinalis. The crude extract was analyzed for antidiabetic activity and subjected to column chromatography. The isolated fractions were screened in vitro for α-glucosidase and α-amylase inhibitory activities. The cytotoxicity of the isolated fractions was studied on L929 cell lines. Following which, the screened fraction 2 was allowed for structure elucidation using gas chromatography-mass spectrometry, one-dimensional, two-dimensional nuclear magnetic resonance spectroscopy, ultraviolet, and Fourier-transform infrared analysis. The binding energies of the isolated fraction 2 with glycolytic enzymes were calculated by molecular docking studies using AutoDock Vina. The isolated fraction 2 identified as 2,4,6-triphenylaniline, showed no significant difference in α-amylase inhibition rates and a significant difference of 10% in α-glucosidase inhibition rates than that of the standard drug acarbose. Further, the cytotoxicity assay of the isolated fraction 2 resulted in a cell viability of 73.96%. Supportingly, in silico studies showed 2,4,6-triphenylaniline to produce a stronger binding affinity toward the glycolytic enzyme targets. The compound 2,4,6-triphenylaniline isolated from A. longipes strain VITN14G exhibited satisfactory antidiabetic activity for type 2 diabetes in vitro, which will further be confirmed by in vivo studies. Successful outcome of the study will result in a natural substitute for existing synthetic antidiabetic drugs.     

Identification of protein targets in red complex organisms binding with resveratrol

Periodontitis is attributed to the dental biofilm formation. Red complex organisms are a group of organisms linked with periodontal diseases. Therefore, it is of interest to identify potential targets from the red complex organisms to bind with the herbal compound resveratrol (E - 5 - (4 - hydroxy styryl) benzene 1,3 diol). We report a list of potential proteins having optimal drug like binding features with the herbal agent Resveratrol for further consideration. We used the STITCH v.5 pipeline VICMPred and VirulentPred tools to identify such targets as potential virulent factors in the red complex organisms. We considered the strains of Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405 and Tannerella forsythia ATCC 43037 in the red complex pathogens for this analysis. Protein targets in the red complex organisms with optimal binding features with the herbal compound resveratrol were thus identified and reported for further consideration.     

Identification and comparative analysis of <Emphasis Type="Italic">Brassica juncea</Emphasis> pathogenesis-related genes in response to hormonal,biotic and abiotic stresses

Pathogenesis-related proteins (PRs) are the antimicrobial proteins which are commonly used as signatures of defense signaling pathways and systemic acquired resistance. However, in Brassica juncea most of the PR proteins have not been fully characterized and remains largely enigmatic. In this study, full-length cDNA sequences of SA (PR1, PR2, PR5) and JA (PR3, PR12 and PR13) marker genes were isolated from B. juncea and were named as BjPR proteins. BjPR proteins showed maximum identity with known PR proteins of Brassica species. Further, expression profiling of BjPR genes were investigated after hormonal, biotic and abiotic stresses. Pre-treatment with SA and JA stimulators downregulates each other signature genes suggesting an antagonistic relationship between SA and JA in B. juncea. After abscisic acid (ABA) treatment, SA signatures were downregulated while as JA signature genes were upregulated. During Erysiphe cruciferarum infection, SA- and JA-dependent BjPR genes showed distinct expression pattern both locally and systemically, thus suggesting the activation of SA- and JA-dependent signaling pathways. Further, expression of SA marker genes decreases while as JA-responsive genes increases during drought stress. Interestingly, both SA and JA signature genes were induced after salt stress. We also found that BjPR genes displayed ABA-independent gene expression pattern during abiotic stresses thus providing the evidence of SA/JA cross talk. Further, in silico analysis of the upstream regions (1.5 kb) of both SA and JA marker genes showed important cis-regulatory elements related to biotic, abiotic and hormonal stresses.