Leptospiral cell wall hydrolase (LIC_10271) binding peptidoglycan, lipopolysaccharide, and laminin and the protein show LysM and M23 domains are co-existing in pathogenic species.

Leptospirosis, a global reemerging zoonosis caused by the spirochete Leptospira, has severe human and veterinary implications. Cell wall hydrolase (LIC_10271) with LytM (peptidase M23) and LysM domains are found to be associated with various pathogenic bacteria. These domains regulate effects on extracellular matrix and biofilm components, which promote cell wall remodeling and pathogen dissemination in the host. In this study, we present the cloning, expression, purification, and characterization of LIC_10271. To determine the localization of LIC_10271 within the inner membrane of Leptospira, Triton X-114 subcellular fractionation and immunoblot studies were performed. Furthermore, r-LIC_10271 binds with peptidoglycan, lipopolysaccharide, and laminin in a dose-dependent manner. Analysis of the signal peptide, M23, and LysM domains revealed conservation primarily within the P1 group of Leptospira, which encompasses the most pathogenic species. Moreover, the presence of native-LIC_10271 in the inner membrane and the distribution of M23 and LysM domains across pathogenic strains indicates their potential involvement in the interaction between the host and Leptospira.

Leptospiral imelysin (LIC_10713) is secretory, immunogenic and binds to laminin, fibronectin, and collagen IV.

Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira. Early and accurate diagnosis is the prime step in managing the disease. Secretory proteins of Leptospira remain distinguished for diagnosis due to their availability as soluble proteins in the serum and their interaction with the host immune response due to their extracellular presence. This study presents the cloning, expression, purification, and characterization of imelysin or LruB (LIC_10713), a putative leptospiral protein. We report that the localization of imelysin showed its presence in the inner membrane and in the culture supernatant. The imelysin was upregulated under in vitro physiological conditions of infection. The LIC_10713 interacted significantly with laminin, fibronectin, collagen type I, and collagen type IV in a dose-dependent manner. Phylogenetic analysis showed that LIC_10713 is predominately found in the pathogenic species of Leptospira, and the GxHxxE motif of imelysin-like proteins is represented as the amino acid sequence GWHAIE. Also, immunoglobulins in leptospirosis-infected patients recognize recombinant-LIC_10713 with 100% specificity and 90.9% sensitivity. The secretion nature, abundance, upregulation, binding to ECM components, and immunogenicity determine LIC_10713 as an important molecule that can be used as an anti-leptospirosis measure. KEY POINTS: • The imelysin-like protein (LIC_10713) of Leptospira is a secretory protein • The protein LIC_10713 can bind ECM molecules • The LIC_10713 is mainly found in pathogenic leptospires • The anti-LIC_10713 antibody from human serum can detect the r-LIC_10713.

Peeping into Mitochondrial Diversity of Andaman Goats: Unveils Possibility of Maritime Transport with Diversified Geographic Signaling.

Andaman and Nicobar Islands, a part of South-East Asia, is enriched with the presence of native breeds of livestock (cattle, pig, goat) and poultry. There are two native goat breeds, viz., Andaman local goat and Teressa goat in Andaman and Nicobar Islands. However, to date, the origin and genetic makeup of these two breeds have not been detailed. Therefore, the present study describes the genetic makeup of Andaman goats through analysis of mitochondrial D-loop sequence for sequence polymorphism, phylogeographical signaling and population expansion events. The genetic diversity of the Teressa goat was less compared to the Andaman local goat due to its sole presence on Teressa Island. Out of 38 well-defined haplotypes of Andaman goats, the majority of haplotypes belonged to haplogroup A followed by haplogroup B and haplogroup D. The result of mismatch distribution and neutrality tests indicated no population expansion event of haplogroup A and B. Finally, based on poor geographical signaling, we hypothesize that Andaman goats have been imported to these Islands either through multidirectional diffusion or unidirectional diffusion. We justify our hypothesis of multidirectional diffusion on the basis of observation of the haplotype and nucleotide diversity of Andaman goats. Simultaneously, the probability of unidirectional diffusion of goats in these islands from the Indian subcontinent in different spells of domestication events through maritime routes cannot be ignored.

Supplementing turmeric rhizome powder in growing Andaman local pigs: a conflated approach for therapy evaluation.

This paper examined the pluripotent effect of supplementation of turmeric rhizome powder (TRP) (Curcuma longa) in growing Andaman local pigs. A total of 48 pigs were randomly allotted into four groups and fed diets containing TRP at 4 concentrations, that is, 0 (control group), 0.05 (treatment 1), 0.1 (treatment 2), and 0.2% (treatment 3) for 30 days. The mean body weight of pigs supplemented with 0.1% and 0.2% TRP was significantly higher than that of the control group (41.66 ± 0.44, 42.59 ± 0.33 vs 40.38 ± 0.30 kg; p ≤ 0.05) which indicated the effect of TRP as growth enhancer. A significant (p ≤ 0.05) decrease in serum concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) was recorded in supplemented groups as compared to the control group. Creatinine kinase (U/l) decreased in all the treatment groups as compared to the control group. Total cholesterol, triglyceride, and low-density lipoprotein cholesterol decreased significantly after supplementation with 0.1% and 0.2% TRP in comparison to the control group. However, there was marked increase in high-density lipoprotein cholesterol (mg/dl) in all TRP-supplemented groups than the control group (27.67 ± 0.60 in T1, 32.76 ± 0.32 in T2, and 34.58 ± 0.37 in T3 vs. 23.73 ± 0.69 in control; p ≤ 0.05). Further, there was increase in antioxidant profile after TRP supplementation. Anti-inflammatory potentiality of TRP could also be appreciated since TRP supplementation downregulated (p ≤ 0.05) expression of IL-6, IL-1ß, and IFN-γ. Therefore, we perceive that this conflated approach is an example of its own kind to focus on modification of health status of pigs for more productivity and augmentation of immune response.

Maternal lineage of Nicobari pig (Sus scrofa nicobaricus) correlated with migration of Nicobarese, a native tribal population of Andaman and Nicobar Islands, India.

Nicobari pig is reared by Nicobarese, a native tribal population of Andaman and Nicobar Islands. Nicobari pig has maintained its genetic identity due to geographical isolation. This communication is the first report on maternal inheritance of Nicobari pigs. DNA polymorphism data showed seven haplotypes. D-loop sequence information and mitogenome analysis were able to earmark Nicobari pigs to Asian clade. The domestication process of pigs and its expansion pattern help to understand human migration pattern. Based on this hypothesis, this communication elucidates the probable origin of Nicobarese. Earlier studies indicated that Nicobarese had genetic affinities to races distributed in China, Malaysia and Thailand. Our data on maternal inheritance of Nicobari pig correlates with the data on migration of Nicobarese. Moreover, we could establish a novel connection of Nicobarese with people of Northeastern parts of India, Philippines and Vietnam through phylogenetic signal and geographical provenance of Nicobari pig. We further concluded that migration of Nicobarese happened during Western route of migration (WRM) ∼4000 years before present. Therefore, we propose one wave hypothesis of peopling of Nicobar based on our study and existence of Ausrtroasiatic language, Mon-Khmer in these islands.

Legacies of domestication, Neolithic diffusion and trade between Indian subcontinent and Island Southeast Asia shape maternal genetic diversity of Andaman cattle.

Andaman cattle is a precious indigenous livestock species endemic to Andaman and Nicobar Islands, India. Till date, origin and genetic makeup of the breed which is warranted for breed conservation is not known. Moreover, the spread of zebu cattle from Indus valley to different parts of Island Southeast Asia (ISEA) is not properly understood. Here, we report the genetic diversity, population structure of Andaman cattle and their evolution in the context of epicentre of zebu domestication and ISEA. High genetic diversity in complete mitochondrial D-loop sequences indicated the ability of the breed to withstand impending climate change. Total 81 haplotypes were detected and all of them except three belonged to Bos indicus. The presence of taurine haplotypes in Andaman cattle indicate introgression by European-derived cattle. A poor phylogenetic signal of Andaman cattle with genetic affinities with cattle of Indian subcontinent and ISEA was observed. The poor phylogenetic structure may be due to multidirectional gene flow from Indian subcontinent and ISEA, with which Andaman shares a close cultural and trade relationship from Neolithic age. We hypothesize that Andaman cattle is the outcome of Neolithic diffusion from centre of zebu domestication along with multidirectional commercial exchange between Indian subcontinent and ISEA.

Molecular characterization of Rhipicephalus microplus in Andaman and Nicobar Islands, India: an insight into genetic assemblages.

The tick, Rhipicephalus microplus is considered as the most notorious ectoparasite of veterinary importance in tropical and sub-tropical regions of the world. The present study deals with the molecular characterization of R. microplus in different regions of Andaman and Nicobar Islands using sequence information of mitochondrial cytochrome C oxidase subunit I (COX1) and their phylogenetic relationship with other Indian R. microplus genotypes. DNA polymorphism study identified a total of eight haplotypes with haplotype diversity of 0.909 ± 0.065 and nucleotide diversity of 0.01911 ± 0.00493. Currently, R. microplus complex consists of five taxa; R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014), R. microplus clade C sensu Low et al. (2015), R. australis and R. annulatus. Phylogenetic analysis revealed the presence of two clades (clade A and clade C) of R. microplus in Andaman and Nicobar isolates; Nicobar isolates belonged to clade A whereas Andaman isolates belonged to clade C of R. microplus complex. All the other Indian sequences retrieved from GenBank belonged to clade C of R. microplus complex. Andaman isolates under clade C of R. microplus were phylogenetically distinct from Indian isolates, which indicates independent speciation under isolated island milieu. In Indian isolates, no host-specific or geographical location-specific sub-clustering was observed which indicates the species jumping potential of the R. microplus tick. Therefore, this study indicated the presence of two different genetic makeup of R. microplus complex in two areas of the Andaman and Nicobar archipelago separated by a natural geographical barrier. This indicates presence of two different founding populations of ticks, one in the south and north-middle Andaman and the other in Nicobar Island.

Genetic structure, population diversity and ancestry of Nicobari fowl based on mtDNA complete D-loop sequences.

Nicobari fowl constitute an endemic poultry germplasm of Andaman and Nicobar Islands, India. Genetic diversity, population structure and ancestry of Nicobari fowl were analysed with mitochondrial D-loop sequences. Analysis of complete D-loop sequences (1231-1232 bp) showed 46 polymorphic sites resulting in 26 haplotypes with overall haplotype diversity of 0.895 and nucleotide diversity of 0.0064. Analysis of molecular variance of spatial populations (sampling sites) of Nicobari fowl revealed that the estimated FST value as 0.229 among the populations. Tajima's D and Fu's FS tests indicated nonsignificant deviation from neutrality and the multimodal pattern of mismatch distribution in demographic expansion suggested that Nicobari fowl populations are in equilibrium. The median-joining (MJ) network of D-loop sequences with reference haplogroup sequences identifies the presence of haplogroups A, B, E1, E2, F and I in Nicobari fowl. The major haplogroup in Nicobari fowl was E (60%), which is otherwise found mainly in the Indian subcontinent. Phylogenetic analysis of Nicobari fowl with junglefowl by maximum likelihood method showed Gallus gallus murghi and G. g. spadiceus as maternal progenitors. Grouping of Nicobari fowl with their primary ancestor, Indian red Junglefowl (G. g. murghi) and the presence of Indian subcontinent-specific haplogroups (E2 and I) support the independent domestication of chickens in India. This study will help to design breeding strategy for conservation of Nicobari fowl in its island habitat.

Deducing Insulin-Producing Cells from Goat Adipose Tissue-Derived Mesenchymal Stem Cells.

Mesenchymal stem cell is a potent tool for regenerative medicine against control of incurable diseases in human and animals. Diabetes mellitus is one such condition marked with the blood glucose is high due to lack of insulin (INS) hormone secreted by the pancreatic cells. Rare, but sporadic, cases of dysfunctional pancreatic cells in goat as well as the promises of stem cell therapy as an off-the-shelf medicine prompted us to explore the potential of adipose-derived goat mesenchymal stem cells (AD-MSCs) to transdifferentiate into pancreatic islet-like cells. We isolated, in vitro cultured, and characterized the AD-MSCs by expression of MSC-specific markers and differentiation into multiple mesodermal lineage cells. The characterized AD-MSCs were in vitro transdifferentiated into INS-producing islet-like cells using a cocktail of glucose, nicotinamide, activin-A, exendin-4, pentagastrin, retinoic acid, and mercaptoethanol in 3 weeks. The transdifferentiated islet-like cells demonstrated the expression of pancreatic endoderm-specific transcripts PDX1, NGN3, PAX6, PAX4, ISL1, and GLUT2 as well as protein expression of pancreatic and duodenal homeobox 1 (PDX1), INS, and Islets 1 (ISL1). The islet-like cells also demonstrated the significant glucose-dependent INS release with respect to the course of transdifferentiation regime. The study envisaged to create the building material for basic research into mechanism of glucose homeostasis, which may pave road for developments in diabetes drug discovery and regenerative therapies.

Exploring broilers and native fowls of Andaman and Nicobar Islands as a source of ß-lactamase-producing Enterobacteriaceae even with limited anthropogenic activities and docking-based identification of catalytic domains in novel ß-lactamase variants.

Objectives: The present study was conducted to detect the occurrence of ß-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the ß-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database. Materials and methods: A total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical ß-lactamase producers were identified by a double-disc test. The genes (bla CTX, bla SHV, bla TEM , and bla AmpC) were screened, and selected sequences of ß-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different ß-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the ß-lactamase variants. The selected ß-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank. Results: In total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61-76%) and the lowest against gentamicin (15-20%). Phenotypical production of ß-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of ß-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the ß-lactamase-producing isolates revealed the maximum possession of bla TEM, followed by bla SHV and bla CTX - M. The nucleotide sequences were found to be similar with bla CTX - M-15, bla SHV - 11, bla SHV - 27, bla SHV - 228, bla TEM - 1, and bla AmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from -8.1 (SHV-27 + cefotaxime) to -7 (TEM-1 + cefotaxime) kcal/mol. Conclusion and relevance: The study revealed ß-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed bla TEM - 1, followed by bla CTX - M-15. Possession of bla SHV - 11, bla SHV - 27, and bla SHV - 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of ß-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Physiological and immune responses to long road transportation in Andaman local pigs.

Transportation of farm animals is a routine management practice. During transportation, animals experience physiological and psychological stress which has negative consequences on health and welfare of animals. Effect of transportation on pig health and welfare in island ecosystem is not properly known. Here, we report the effect of transportation stress under island ecosystem on serum biochemical parameters, lipid profile, stress biomarkers, and immune parameters in Andaman local pig. Twelve pigs were transported from North and Middle Andaman to South Andaman via road in which two small sea channels have to be crossed in ferry with vehicle on board. Transportation triggered increase in serum levels of total protein and albumin. Elevated levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and creatine kinase in transported pigs indicated muscular exercise or impaired liver function. Transportation induced dyslipidemia indicated by elevated levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides and a decreased level of high-density lipoprotein cholesterol. High level of serum total nitric oxide and serum MDA as well as low antioxidant capacity in transported pigs indicated oxidative stress induction. Transportation resulted in activation of stress response and increased levels of serum cortisol and heat shock proteins were recorded in transported pigs. Moreover, increased concentration of pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-1ß, and IFN-γ) and decreased concentration of anti-inflammatory cytokine IL-4 were detected in transported pigs. The results of the study are helpful for better understanding of transportation stress in pigs under island ecosystem.

Tri-Model Therapy: Combining Macrocyclic Lactone, Piperazine Derivative and Herbal Preparation in Treating Humpsore in Cattle.

Stephanofilariasis or humpsore is a chronic parasitic dermatitis of cattle. Various treatment regimens were attempted in the past but were found to be partially effective. Here, we claim a successful treatment regime using an FDA-approved macrocyclic lactone, a piperazine derivative, and an herbal preparation. Twenty-four cattle (18 affected and 6 unaffected) were selected and divided into Gr 1: positive control (infected without treatment; n = 6), Gr 2: treatment group (infected with treatment with ivermectin; n = 6), Gr 3: treatment group (infected with treatment with tri-model therapy including ivermectin, diethylcarbamazine citrate, and an herbal ointment, n = 6), and Gr 4: negative control (non-infected animals; n = 6). In Gr 2 and Gr 3, treatment to the ailing animals were given for 30 days. Lesion was significantly reduced in day 15 of post-treatment and completely healed on day 30 of post-treatment in Gr 3. Tri-model therapy recorded significant improvement in the surface area of the sore as compared to ivermectin administration alone. Antioxidants were increased and malondialdehyde (MDA) and cortisol concentrations were decreased significantly (p < 0.05) in Gr 3 than in untreated control group at day 14, 21 and 28. Histopathological changes in infected animals were characterized by parakeratotic hyperkeratosis along with presence of nucleated keratinocytes. There were infiltrations of polymorphonuclear cells specially eosinophils along with a few monomorphonuclear cells. Microfilarial organism was observed beneath the epidermis, which was surrounded by fibrocytes and infiltrated cells. In the tri-model-treated animal after recovery, the skin revived a normal architecture. Therefore, tri-model therapy has the potential to cure humpsore.

Origin, genetic diversity and evolution of Andaman local duck, a native duck germplasm of an insular region of India.

Domestic ducks are of paramount importance as a cheap source of protein in rural India. Andaman local duck (ALD) is an indigenous avian genetic resource of Andaman and Nicobar islands (ANI) and is mainly distributed in Middle and Northern parts of these islands. Negligence has brought this breed on the edge of extinction necessitating immediate conservation efforts. Here, we report the genetic diversity, population structure and matrilineal genetic root of ALD. Partial mtDNA D-loop sequences were analyzed in 71 ALD samples and analysis revealed 19 polymorphic sites and 13 haplotypes. Estimated haplotype (Hd ± SD) and nucleotide diversity (π ± SD) were 0.881 ± 0.017 and 0.00897 ± 0.00078 respectively. The high genetic diversity of ALD indicates introgression of genetic material from other local duck breeds. In addition, it can be postulated that ALD bearing high genetic diversity has strong ability to adapt to environmental changes and can withstand impending climate change. Phylogenetic and network analysis indicate that ALD falls under Eurasian clade of mallard and ALD forms three clusters; one cluster is phylogenetically close to Southeast Asian countries, one close to Southern part of mainland India and the third one forms an independent cluster. Therefore, ALD might have migrated either from Southeast Asian countries which enjoy a close cultural bondage with ANI from time immemorial or from Southern part of India. The independent cluster may have evolved locally in these islands and natural selection pressure imposed by environmental conditions might be the driving force for evaluation of these duck haplotypes; which mimics Darwin's theory of natural selection. The results of the study will be beneficial for formulating future breeding programme and conservation strategy towards sustainable development of the duck breed.

Host-Parasite Interaction in Sarcoptes scabiei Infestation in Porcine Model with a Preliminary Note on Its Genetic Lineage from India.

The burrowing mite Sarcoptes scabiei causes scabies in humans or mange in animals. It infests a wide range of mammalian species including livestock, companion animals, wild animals, and humans. Differential diagnosis of Sarcoptes varieties is key for epidemiological studies and for formulation of an eradication program. Host-parasite interaction at the systemic level is very important to understand the pathogenicity of the mite. This communication deals with the preliminary report on the genetic characterization of S. scabiei from India. Moreover, the effect of S. scabiei infestation on host physiology with special emphasis on serum biochemical parameters, lipid profile, oxidant/antioxidant balance, stress parameters, and immune responses were evaluated in a porcine model. Cytochrome C oxidase 1 and voltage-sensitive sodium channel based phylogenetic study could distinguish human and animals isolates but could not distinguish host or geographical specific isolates belonging to animal origin. An absence of host-specific cluster among animal isolates argues against the hypothesis of delineating S. scabiei as per host origin. Elevated levels of markers of liver function such as albumin, AST, ALT, ALP, and LDH in infested animals indicated impaired liver function in infested animals. S. scabiei infestation induced atherogenic dyslipidemia indicated by elevated levels of total cholesterol, low-density lipoprotein cholesterol and triglycerides, and a decreased level of high-density lipoprotein cholesterol. Oxidative stress in infested animals was indicated by a high level of nitric oxide and serum MDA as oxidative stress markers and low antioxidant capacity. S. scabiei triggered stress response and elevated levels of serum cortisol and heat shock proteins were recorded in infested animals. S. scabiei infestation increased the serum concentration of immunoglobulins and was associated with up-regulation of IL-2, IFN-γ, IL-1ß, and IL-4 indicating both Th1 and Th2 response. The results of the study will be helpful for a better understanding of host-parasite interaction at the systemic level in crusted scabies in pigs.

Tracing the genetic root of Trinket cattle: an endangered cattle population of a small isolated island of Nicobar group of islands.

Trinket cattle are the inhabitant of a small island called Trinket, which is one of the picturesque islands of Andaman. This herd is thought to be of Danish leftover during their dynasty in Nicobar archipelago. When the island was abandoned by foreign invaders, indigenes utilized the animals for the purpose of meat. As a result, the cattle became semi-feral in nature. After the Great Sumatra earthquake and tsunami of Indian Ocean in 2004, Trinket island was left abandoned by indigenes and the cattle became totally feral in nature. To trace the genetic root of the cattle, this study has been undertaken based on the sequence information of the mitochondrial D-loop and cytochrome b gene. The genomic DNA was extracted from the blood samples of the Trinket cattle and was used for amplification of mitochondrial markers, and the sequence information was generated by Sanger sequencing. The analysis of sequence information revealed that the Trinket cattle belongs to Bos indicus (I) haplotype, sub-haplotype I2. The presence of I2 sub-haplotype in Trinket cattle may be due to the expansion of this I2 haplotype towards Southeast Asian countries. This is a novel input for the formulation of breeding strategy towards conservation of eco-friendly sustainable livestock in the isolated island ecosystem.

Exogenous cholesterol prevents cryocapacitation-like changes, membrane fluidity, and enhances in vitro fertility in bubaline spermatozoa.

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+ , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+ ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+ , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+ , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.

A Natural Quinazoline Derivative from Marine Sponge Hyrtios erectus Induces Apoptosis of Breast Cancer Cells via ROS Production and Intrinsic or Extrinsic Apoptosis Pathways.

Here, we report the therapeutic potential of a natural quinazoline derivative (2-chloro-6-phenyl-8H-quinazolino[4,3-b]quinazolin-8-one) isolated from marine sponge Hyrtios erectus against human breast cancer. The cytotoxicity of the compound was investigated on a human breast carcinoma cell line (MCF-7). Antiproliferative activity of the compound was estimated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay showed significant inhibition of MCF-7 cells viability with the IC50 value of 13.04 ± 1.03 µg/mL after 48 h. The compound induced down-regulation of anti-apoptotic Bcl-2 protein and increase in the pro-apoptotic Bax/Bcl-2 ratio in MCF-7 cells. The compound activated the expression of Caspases-9 and stimulated downstream signal transducer Caspase-7. In addition, Caspase-8 showed remarkable up-regulation in MCF-7 cells treated with the compound. Moreover, the compound was found to promote oxidative stress in MCF-7 cells that led to cell death. In conclusion, the compound could induce apoptosis of breast carcinoma cells via a mechanism that involves ROS production and either extrinsic or intrinsic apoptosis pathways. The systemic toxic potential of the compound was evaluated in an in vivo mouse model, and it was found non-toxic to the major organs.

Effect of Prophylactic Management of Hemophilia on Bleeding Episodes.

Hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency) are the most common and serious congenital coagulation factor deficiencies with repeated hemarthroses leading to development of target joints. Continuous prophylaxis is regular infusion of factor concentrates at fixed dose at regular interval to prevent hemorrhages. The study was designed to assess the outcome of continuous prophylaxis in hemophilia on bleeding episodes. It was conducted from November, 2017 to April, 2018 in the Paediatrics Department of Midnapore Medical College, Paschim Medinipur, West Bengal on 33 boys from 4 to 18 years of age suffering from Hemophilia with frequent bleeding episodes. Prior to starting continuous prophylaxis all the patients' target joint(s) were assessed based on Gilbert Score and bleeding episodes in the last 6 months were assessed based on Annualized Bleeding Rate and ISTH-BAT Score. All the children were provided prophylaxis therapy with plasma derived Factor concentrate twice per week at a dose of 20 + - 2 IU/Kg. All bleeding episodes of the children during prophylaxis were recorded. Bleeding episodes showed significant improvement only in children who could sufficiently adhere to continuous prophylactic therapy. Continuous prophylaxis reduces bleeding episodes in Hemophilia in twice weekly protocol provided the patients sufficiently adhere to continuous prophylaxis regimen.

Mitogenome analysis of Indian isolate of Rhipicephalus microplus clade A sensu (): A first report from Maritime South-East Asia.

This communication reports a comprehensive profile of mitogenome analysis of Rhipicephalus microplus, isolated and identified from Andaman and Nicobar islands, a part of Maritime South East Asia. Complete mitogenome of Indian isolate of R. microplus (MK234703) was 14,903 bp. Mitochondrial (mt.) genome had 13 protein coding genes (PCGs), 22 tRNAs, two ribosomal subunits and two control regions. All PCGs were located on the H-strand except nad1, nad5, nad4 and nad4L. All start codons were ATN codon and abbreviated stop codons were seen in cox-2-3, nad-5 and cytb. A purine rich tick-box motif has been identified. A tandem repeat unit (TTTATT), described as a region alike to nad1 was identified in 130 bp insertion in between nad1 and tRNA-Glu and in nad1 sequence. Presence of two control regions (CRs) proved that, two CRs have evolved in concert rather than independently. Strong biasness towards A and T in Indian isolate of R. microplus is a typical feature for most of the arthropods. Subtracted values of dn and ds suggested that, there was least effect of nt. sequence of cox1 gene when Indian isolate was compared with other isolates of Rhipicephalus. On the basis of phylogenetic analysis, species of the genus Rhipicephalus could be clustered in three groups; ticks of the genera belonging to sub-family Rhipicephalinae could be grouped in a single cluster. Finally, cox1 sequence of MK234703 indicated that the isolate belonged to clade A sensu Burger et al., 2014 which has not been reported earlier from India.

Variability of the EG95 antigen-coding gene of Echinococcus granulosus in animal and human origin: implications for vaccine development.

In the present study, the genetic variability of the EG95 protein-coding gene in several animal and human isolates of Echinococcus granulosus was investigated. A total of 24 isolates collected from cattle, buffalo, sheep, goat, dog and man were amplified by Eg95-coding gene-specific primers. From the generated sequence information, a conceptual amino acid sequence was deduced. Phylogenetically, the Eg95 coding gene belongs to the Eg95-1/Eg95-2/Eg95-3/Eg95-4 cluster. Further confirmation on the maximum composite likelihood analysis revealed that the overall transition/transversion bias was 2.913. This finding indicated thatthere is bias towards transitional and transversional substitution. Using artificial neural networks, a B-cell epitope was predicted on primary sequence information. Stretches of amino acid residues varied between animal and human isolates when hydrophobicity was considered. Flexibility also varied between larval and adult stages of the organism. This observation is important to develop vaccines. However, cytotoxic T-lymphocyte epitopes on primary sequence data remained constant in all isolates. In this study, agretope identification started with hydrophobic amino acids. Amino acids with the same physico-chemical properties were present in the middle. The conformational propensity of the Eg95-coding gene of 156 amino acid residues had α-turns and ß-turns, and α-amphipathic regions up to 129, 138-156 and 151-155 residues, respectively. The results indicated potential T-cell antigenic sites. The overall Tajima's D value was negative (-2.404165), indicative of negative selection pressure.