Genomic insights into an endophytic Streptomyces sp. VITGV156 for antimicrobial compounds.

Endophytic Streptomyces sp. are recognized as a potential resource for valuable natural products but are less explored. This study focused on exploring endophytic Streptomyces species residing within tomato plants (Solanum lycopersicum) harboring genes for the production of a novel class of antibiotics. Our research involved the isolation and characterization of Streptomyces sp. VITGV156, a newly identified endophytic Streptomyces species that produces antimicrobial products. VITGV156 harbors a genome of 8.18 mb and codes 6,512 proteins, of which 4,993 are of known function (76.67%) and 1,519 are of unknown function (23.32%). By employing genomic analysis, we elucidate the genome landscape of this microbial strain and shed light on various BGCs responsible for producing polyketide antimicrobial compounds, with particular emphasis on the antibiotic kendomycin. We extended our study by evaluating the antibacterial properties of kendomycin. Overall, this study provides valuable insights into the genome of endophytic Streptomyces species, particularly Streptomyces sp. VITGV156, which are prolific producers of antimicrobial agents. These findings hold promise for further research and exploitation of pharmaceutical compounds, offering opportunities for the development of novel antimicrobial drugs.

Blue-Light-Activated Water-Soluble Sn(IV)-Porphyrins for Antibacterial Photodynamic Therapy (aPDT) against Drug-Resistant Bacterial Pathogens.

Antimicrobial resistance has emerged as a global threat to the treatment of infectious diseases. Antibacterial photodynamic therapy (aPDT) is a promising alternative approach and is highly suitable for the treatment of cutaneous bacterial infections through topical applications. aPDT relies on light-responsive compounds called photosensitizer (PS) dyes, which generate reactive oxygen species (ROS) when induced by light, thereby killing bacterial cells. Despite several previous studies in this area, the molecular details of targeting and cell death mediated by PS dyes are poorly understood. In this study, we further investigate the antibacterial properties of two water-soluble Sn(IV) tetrapyridylporphyrins that were quaternized with methyl and hexyl groups (1 and 2). In this follow-up study, we demonstrate that Sn(IV)-porphyrins can be photoexcited by blue light (a 427 nm LED) and exhibit various levels of bactericidal activity against both Gram-(+) and Gram-(-) strains of bacteria. Using localization studies through fluorescence microscopy, we show that 2 targets the bacterial membrane more effectively than 1 and exhibits comparatively higher aPDT activity. Using multiple fluorescence reporters, we demonstrate that photoactivation of 1 and 2 results in extensive collateral damage to the bacterial cells including DNA cleavage, membrane damage, and delocalization of central systems necessary for bacterial growth and division. In summary, this investigation provides deep insights into the mechanism of bacterial killing mediated by the Sn(IV)-porphyrins. Moreover, our approach offers a new method for evaluating the activity of PS, which may inspire the discovery of new PS with enhanced aPDT activity.

Agreement and repeatability of Icare ic100 tonometer.

PURPOSE: To find the agreement and repeatability of Icare ic100 tonometer. METHODS: We included 150 subjects above the age of 18 years for this cross-sectional, multicenter study with intraocular pressure (IOP) ≥7 mmHg. After the initial ophthalmic examination, two masked examiners took five IOP measurements using three different instruments; Icare ic100, Icare TA01i, and Goldmann applanation tonometer (GAT) in only one eye of the participants. Comparison of agreement of IOP using different instruments was quantified with intraclass correlation coefficient (ICC) using the two-way random effects models of absolute agreement and Cronbach's alpha. The test-retest variability of the instruments was assessed by deriving repeatability coefficient (RC) and coefficient of variation (CV). RESULTS: Agreement between the tonometers across the different IOP groups had no statistically significant difference in their mean IOP. Icare ic100 was found to have good reliability across all IOP groups (ICC value >0.78) when compared with Icare TA01i. In comparison with GAT, Icare ic100 showed good reliability across all IOP groups (ICC >0.87) except >16 to <23 mmHg group where it showed moderate reliability (ICC = 0.52). Icare ic100 showed good repeatability with RC and CV of 2.67 and 4.89, respectively. CONCLUSION: Icare ic100 rebound tonometer can measure IOP with relatively small measurement error and can provide a reliable and repeatable reading in comparison with GAT across a wide pressure range without hampering corneal health.

A Weighted Speaker-Specific Confusion Transducer-Based Augmentative and Alternative Speech Communication Aid for Dysarthric Speakers.

An augmentative and alternative speech communication (AASC) aid comprises a speech recognition system and a speech synthesis system. The main challenge in developing such an aid for dysarthric speakers lies in handling errors in the text derived from the recognition system. These errors (substitution, deletion, and insertion) may be due to inability of a dysarthric speaker to utter certain phones (articulatory error) or due to inaccuracy of the models trained (modeling error). Most existing AASC approaches only focus on the articulatory errors and the ones that do address both errors, and do not differentiate between them. However, this paper performs a three-level cascaded analysis to identify and distinguish between these errors, as differentiating these errors will aid in appropriately handling them. Furthermore, analyses in the paper are independent of the syntax of utterances. Based on these analyses, weighted phone confusion transducers are formulated and used to correct erroneous text from the recognition system. The corrected text is finally synthesized by a text-to-speech synthesis system. The proposed AASC is observed to significantly reduce a word error rate of severe dysarthric speakers from 100% to 41.52%, moderate from 61.85% to 18.08%, and mild from 12.23% to 8.55%.

Characterization of a novel natural cellulosic fiber from Prosopis juliflora bark.

Natural fibers from plants are ideal choice for producing polymer composites. Bark fibers of Prosopis juliflora (PJ), an evergreen plant have not been utilized for making polymer composites yet. Hence, a study was undertaken to evaluate their suitability as a novel reinforcement for composite structures. PJ fiber (PJF) was analyzed extensively to understand its chemical and physical properties. The PJF belonged to gelatinous or mucilaginous type. Its lignin content (17.11%) and density (580 kg/m(3)) were relatively higher and lower, respectively in comparison to bark fibers of other plants. The free chemical groups on it were studied by FTIR and XRD. It had a tensile strength of 558±13.4 MPa with an average strain rate of 1.77±0.04% and microfibril angle of 10.64°±0.45°. Thermal analyses (TG and DTG) showed that it started degrading at a temperature of 217 °C with kinetic activation energy of 76.72 kJ/mol.

Phylogenetic analysis of nucleoprotein gene of dog rabies virus isolates from Southern India.

India like several other South East Asian and African countries continues to face the public health and economic problems associated with the disease. Our objective was to perform a limited sequence analysis of a portion of nucleoprotein gene of 22 rabies virus isolates obtained from domestic animals in Southern India during 2004-2005. These isolates were compared with rabies virus isolates originating from Asia, Europe, Africa and North America. The phylogenetic analysis showed that RV isolates in Southern India belong to genotype 1. They were similar to one another forming a single major genetic cluster not ordered by geography or species of origin. However, they were dissimilar to RV isolates in Northern India and in other parts of the world. The data indicated that dog rabies virus variants are the major circulating viruses and control of dog rabies would result in overall reduction in the burden and incidence of rabies in India.

Identification of cross-contaminated animal cells by PCR and isoenzyme analysis.

Animal cell lines have become very popular substrates for the production of vaccines and biopharmaceuticals. Characterization of candidate production cell lines is central to ensure product safety and maintenance of consistency in the manufacture of biologicals. Nested PCR and isoenzyme analysis have been used widely to prove the identity and purity of various cell lines and primary cells individually and also after deliberate cross-contamination. The nested PCR based on the Cytochrome b (Cyt b) gene of mitochondrial DNA (Mt DNA) was found to be more sensitive than isoenzyme analysis in detecting low levels of contaminants (as low as 1%). Interestingly, competition between different co-cultured cell lines has shown in one case that cross-contamination need not always results in a mixed cell population. The nested PCR technique for the Cyt b gene described in this study appears to be a potential replacement for isoenzyme analysis and here we demonstrate the PCR method used is sensitive and reliable for cell line authentication in a simple, rapid and reliable format to help assure the authenticity of cell substrates for the production of safe vaccines and biopharmaceuticals.

Human monoclonal antibody and vaccine approaches to prevent human rabies.

Rabies, being a major zoonotic disease, significantly impacts global public health. It is invariably fatal once clinical signs are apparent. The majority of human rabies deaths occur in developing countries. India alone reports more than 50% of the global rabies deaths. Although it is a vaccine-preventable disease, effective rabies prevention in humans with category III bites requires the combined administration of rabies immunoglobulin (RIG) and vaccine. Cell culture rabies vaccines have become widely available in developing countries, virtually replacing the inferior and unsafe nerve tissue vaccines. Limitations inherent to the conventional RIG of either equine or human origin have prompted scientists to look for monoclonal antibody-based human RIG as an alternative. Fully human monoclonal antibodies have been found to be safer and equally efficacious than conventional RIG when tested in mice and hamsters. In this chapter, rabies epidemiology, reservoir control measures, post-exposure prophylaxis of human rabies, and combination therapy for rabies are discussed. Novel human monoclonal antibodies, their production, and the significance of plants as expression platforms are emphasized.

Molecular epidemiology of rabies virus isolates in India.

In India, rabies is enzootic and is a serious public health and economic problem. India has a large population of stray dogs which, together with a lack of effective control strategies, might have led to the persistence of rabies virus (RV) in the canine population. Our objective was to study the molecular epidemiology of RV isolates in India based on nucleotide sequence analysis of 29 RV isolates originating from different species of animals in four states. Here we have analyzed two sets of sequence data based upon a 132-nucleotide region of the cytoplasmic domain (CD) of the G gene (G-CD) and a 549-nucleotide region (Psi-L) that combines the noncoding G-L intergenic region (Psi) and a fragment of the polymerase gene (L). Phylogenetic analysis revealed that the RV isolates belong to genotype 1 and that they were related geographically but were not related according to host species. Five different genetic clusters distributed among three geographical regions were identified. Comparison of the deduced amino acid sequences of G-CD between RV isolates revealed three amino acid changes (amino acid 462G [aa462G], aa465H, and aa468K) that distinguished the Indian RVs from RV isolates in other parts of the world. Analysis of the data indicated that the dog rabies virus variants are the major circulating viruses in India that transmit the disease to other domestic animals and humans as well.

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus.

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.