Implications of deltamethrin on hematology, cardiac pathology, and gene expression in Nile tilapia (Oreochromis niloticus) and its possible amelioration with Shatavari (Asparagus racemosus).

Deltamethrin (DM) is one of the extensively used pyrethroids for controlling ectoparasites. Unfortunately, DM is highly toxic to fish as it primarily targets the sodium channels of the plasma membrane thereby affecting their cardiac and nervous systems. The present study investigated the protective efficacy of Shatavari (Asparagus racemosus) against DM-induced cardiotoxicity in Nile tilapia (Oreochromis niloticus). The fish were segregated into nine groups having 36 fish/group maintained in triplicates exposed to DM (1 µg/L) and fed with a diet containing three different concentrations (10 g, 20 g, and 30 g/kg feed) of aqueous extract of A. racemosus (ARE) for 21 days. DM caused significant alterations in the blood and serum parameters, and expression of cardiac and apoptotic genes compared to the control group. The ARE cotreatment significantly reduced the increase in serum transaminases, creatine kinase, and lactate dehydrogenase levels induced by DM. ARE facilitated the regain of electrolyte (sodium, potassium, chloride) homeostasis and antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, and glutathione in DM-exposed fish. The cardiac histology exhibited loose separation of the cardiomyocytes and myofibrillar loss in the DM group which was ameliorated in the DM-ARE cotreatment group. Significant modulations were observed in the expression of cardiac-specific genes (gata4, myh6, tnT, cox1) and apoptosis signaling genes and proteins (HSP70, bax, bcl-2, caspase3), in the cotreatment group compared to the DM-exposed group. The current study suggests that ARE possesses potential cardioprotective properties that are effective in mitigating the toxic effects induced by DM via ameliorating oxidative stress, electrolyte imbalance, and apoptosis in tilapia.

Exploration of clinical and genetic findings in Ataxia-Telangiectasia (AT) patients from the Indian subcontinent.

BACKGROUND: Ataxia-Telangiectasia (AT) is a rare autosomal recessive neurodegenerative disorder. It is caused by mutations in the Ataxia-Telangiectasia mutated (ATM) gene, which codes for protein ATM serine/threonine kinase. OBJECTIVE: We aim to describe the clinical and radiological findings in children and adolescents of 20 molecularly confirmed cases of AT. We aim to correlate these findings with the genotype identified among them. METHODS: This retrospective study included 20 patients diagnosed clinically and genetically with AT over 10 years. The clinical, radiological and laboratory data were extracted from the hospital's electronic medical records. Molecular testing was done using next generation sequencing and Sanger sequencing. In silico predictions were performed for the variants identified by applying Cryp-Skip, Splice site prediction by Neural Network, Mutation Taster and Hope prediction tool. RESULTS: Consanguinity was documented in nearly half of the patients. Telangiectasia was absent in 10%. Microcephaly was seen in 40% cases. The incidence of malignancy in our study population was low. Molecular testing done in the 18 families (20 patients) identified 23 variants of which ten were novel. Biallelic homozygous variants were noted in 13 families and compound heterozygous in 5 families. Out of the 13 families who were homozygous, 8 families (61.5%) (9 patients) have history of consanguinity. In silico prediction of novel missense variants, NM_000051.4 (ATM_v201): c.2702T > C showed disruption of the α-helix of ATM protein and NM_000051.4 (ATM_v201): c.6679C > G is expected to disturb the rigidity of protein structure in the FAT domain. The four novel splice site variants and two intronic variants result in exon skipping as predicted by Cryp-Skip. CONCLUSIONS: AT should be confirmed by molecular testing in young-onset cerebellar ataxia, even without telangiectasia. Awareness of this rare disease will facilitate study of larger cohorts from Indian population to characterize variants and determine its prevalence in this population.

Dietary coriander (Coriandrum sativum L) oil improves antioxidant and anti-inflammatory activity, innate immune responses and resistance to Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

The use of essential oils (EOs) as a natural alternative to antibiotics for disease prevention strategies is gaining much interest in recent decade. Coriander (Coriandrum sativum L.) essential oil is rich in bioactive compounds like linalool and geranyl acetate which have antioxidant, anti-inflammatory and antimicrobial activities. The present work was proposed to evaluate the inclusion levels of coriander oil in tilapia feed to enhance tilapia health and resistance to bacterial infection. Five iso-nitrogenous and iso-lipidic feeds were prepared with graded levels of coriander oil (0, 0.5, 1, 1.5 and 2%). The fish were then fed with the five experimental diet twice daily for a period of 60 days in triplicate. Haemoglobin, mean corpuscular volume, mean cell haemoglobin increased significantly in the coriander oil treated groups. The thrombocyte count was more in 2% inclusion level. The superoxide dismutase activity increased significantly in all the treated groups. The feeds with 1.5 and 2% coriander oil showed increased respiratory burst and myeloperoxidase activities while lysozyme and antiprotease activities were significantly higher in 1, 1.5 and 2% dietary treatments compared to control. The survival increased in dose dependent manner post challenge with an intraperitoneal injection of Aeromonas hydrophila at a LD50 dose of 5 × 106 cfu mL-1. The feed containing 1, 1.5 and 2% of coriander oil showed 89, 100 and 100% survival respectively compared to 39% in control diet. The expression level of IgM and IL-8 increased significantly post challenge with A. hydrophila in coriander oil fed groups. The expressions of TNFα, IL-1ß, TGFß and HSP 70 genes, however, decreased significantly in the treated groups compared to control. Histopathological examination of spleen showed large melano-macrophage centers in control and 0.5% coriander fed group with signs of necrosis and vacuolation post A. hydrophila infection, whereas 1, 1.5 and 2% treated groups showed normal architecture of spleen. From the above observations it can be concluded that coriander oil with 1% incorporation in feed improves tilapia health and resistance to bacterial infection.

Case report of a rare purine synthesis disorder due to 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICAR) deficiency.

BACKGROUND: AICA (5-aminoimidazole-4-carboxamide) ribosiduria is an inborn error in purine biosynthesis caused due to biallelic pathogenic variants in the 5-aminoimidazole-4-carboxamide ribonucleotide-formyltransferase/imp cyclohydrolase (ATIC) gene located on chromosome 2q35. ATIC codes for a bifunctional enzyme, AICAR transformylase and inosine monophosphate (IMP) cyclohydrolase, which catalyse the last two steps of de novo purine synthesis. This disorder has been previously reported in only 4 cases worldwide, and herein, we report the first from India. CASE REPORT: The proband presented with global developmental delay, developmental hip dysplasia (DDH), acyanotic heart disease and nystagmoid eye movements. Whole exome sequencing (WES) identified compound heterozygous pathogenic variants in the ATIC. A novel splice site variant; c.1321-2A > G and a previously reported missense variant; c.1277A > G (p.Lys426Arg) were identified. Segregation analysis of parents showed the father to be a heterozygous carrier for the splice site variant and the mother, a heterozygous carrier for the missense variant. CONCLUSION: This case of a rare genetic disorder of purine biosynthesis of ATIC deficiency is the first case reported from India. Early diagnosis lead to early interventional therapy and genetic counselling.

ß-Defensin: An adroit saviour in teleosts.

ß-Defensin (BD) is an important first line innate defense molecule with potent antimicrobial and immunomodulatory activities in fish. The signatures of ß-defensins are the presence of a net cationic charge and three intramolecular disulfide bonds mediated by six conserved cysteines. It consists of three exons and two introns. The signal peptide is usually conserved and sequence divergence is mostly seen in mature peptide region. The diverse amino acid sequences of matured peptide contribute to a strong positive selection and broad-spectrum antimicrobial activity. It is constitutively expressed in both mucosal as well as systemic sites. Increased expression of ß-defensin was mostly reported in bacterial and viral infections in fish. Its role during parasitic and fungal infections is yet to be investigated. ß-Defensin isoforms such as BD-1, BD-2, BD-3, BD-4 and BD-5 can be witnessed even in early developmental days to different pathogenic exposure in fish. ß-Defensins possess adjuvant properties to enhance antigen-specific immunity promoting both cellular and humoral immune response. It significantly reduces/increases bacterial colonization or viral copy numbers when overexpressed/knockdown. Based on its chemotactic and activating potentials, it can contribute to both innate and adaptive immune responses. With mediated expression, it can also control inflammation. It is potent governing resistance in early developmental days as well. Its expression in pituitary and testis suggests its participation in reproduction and endocrine regulation in fish. Overall, ß-defensins is an important member of antimicrobial peptides (AMPs) with multifunctional role in general homeostasis and to pathogen exposure possessing tremendous therapeutic approaches.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine.

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 µl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

Cloning and characterization of linker histone H1 gene in rohu, Labeo rohita.

Linker histone H1 (LHH1) is an abundant nuclear protein that condenses chromatin to form higher-order structure. The present study reported cloning and sequencing of 942 bp of LHH1 from liver tissue of rohu, Labeo rohita, with a complete coding sequence of 792 bp of having 263 amino acids. The phylogenetic tree of L. rohita LHH1 (LrLHH1) shared maximum similarity with that of Carassius auratus. The three dimensional model and domain architecture of LrLHH1 protein was also predicted using Swiss-Prot and SMART domain software. The expression of LHH1 during ontogeny showed significantly higher transcript level in milt, unfertilized eggs and up to 3 h post-fertilization followed by a dramatic decrease thereafter. The tissue-specific expression showed constitutive expression of LrLHH1 in all examined tissues. The expression of LHH1 during different infection models, namely, bacteria (Aeromonas hydrophila); ectoparasite (Argulus siamensis) and poly I:C induction revealed modulation in the level of expression at varied time points post-exposure in the liver and anterior kidney tissues of rohu. However, a synthetic peptide derived from LHH1 sequence of rohu did not have any detectable antibacterial activity. The present study provided necessary information on the role of this protein during ontogeny and innate immunity in Indian major carp species.

Suitable ratio of dietary L-carnitine and α-ketoglutarate improves growth and health performance in Nile tilapia, Oreochromis niloticus.

L-carnitine (LC) and α-Ketoglutarate (AKG) are important growth promoters used in aquafeed. The study aimed to evaluate the incorporation of LC and AKG at different ratios in the diet of tilapia (initial weight 1.38 ± 0.03 g) in order to facilitate lipid utilization and protein synthesis. Fish were fed six isonitrogenous (~ 30 g/100 g CP) and isolipidic (~ 6 g/100 g CL) diets containing graded LC/AKG ratios of 0 (Control), 0.11, 0.42, 1.00, 2.33 and 9.00 in six treatments for 60 days. Fish fed with LC/AKG ratios 2.33 and 9.00 showed significantly (P < 0.05) higher percentage weight gain, specific growth rate and protein efficiency ratio. Feed conversion ratio in fish-fed diets with LC/AKG ratio 9.00 improved significantly (p < 0.05) than other treatments. The whole-body protein content of tilapia and digestive enzyme activity were significantly higher with higher weight gain. The body lipid content was significantly lower in the LC/AKG ratio 9.00. The liver antioxidant parameters and activity of the immune components were significantly higher in the LC/AKG ratio 9 group. The lower serum triglyceride and cholesterol level was also recorded in LC/AKG ratio 9 group. The histology of the intestine and liver showed increased villi area and decreased lipid droplets, respectively, in tilapia fed with higher LC/AKG ratios. It was concluded from the above results that the higher LC and lower AKG (LC/AKG ratio 9.00) combination attributed maximum lipid utilization and higher protein efficiency and thus better growth performance in tilapia. This was also reflected in activity of digestive enzymes, antioxidant enzymes and immune status in tilapia.

Immune gene expression in cyprinid herpesvirus-2 (CyHV-2)-sensitized peripheral blood leukocytes (PBLs) co-cultured with CyHV-2-infected goldfish fin cell line.

Goldfish is one of the preferred ornamental fish which is highly susceptible to cyprinid herpesvirus-2 (CyHV-2) infection. The present study aimed to analyse immune gene expression in a co-culture of CyHV-2-sensitized goldfish peripheral blood leukocytes (PBLs) with CyHV-2-infected fantail goldfish fin cell lines (FtGF). Goldfish were sensitized with intraperitoneal TCID50 dose (107.8±0.26/mL) of CyHV-2. After 2 weeks, PBLs were collected and co-cultured with CyHV-2-infected FtGF cells keeping both uninfected FtGF cells and PBL control groups. After 2 days of co-culture, WST-1 assay for cell proliferation was performed at 450 nm during the 2nd, 4th and 6th days of co-culture. The results showed a significant increase (p < 0.05) in cell density in CyHV-2-infected PBL and virus-infected FtGF cells during the 4th day post co-culture which confirmed effector cell generation. Expressions of few immune genes were checked taking RNA samples of CyHV-2-induced PBLs post co-culture with infected FtGF cells along with uninfected FtGF cells as control group at different time periods (2nd, 4th and 6th days) in triplicate. The results indicated increased expression of CD8α, IFNγ, b2m, MHC I, LMP 7, IL-10, IL-12 and GATA3 except Tapasin. From the above study, we concluded that goldfish showed both Th1- and Th2-mediated immune responses to CyHV-2. The current findings support the scope for further vaccine development against CyHV-2 for goldfish.

Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish.

The accuracy of quantitative real time PCR (RTqPCR) can be attained only when a suitable reference gene is used. The gene expression for a particular gene may vary within different cells at different conditions. Hence, the suitability and stability of various potential reference genes have to be determined for expression studies. In this study, we have examined the potential of four different reference genes including ß-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3P-dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1AA) in seven different tissues including gill, liver, kidney, spleen, heart, muscle and intestine of goldfish (Carassius auratus). The housekeeping genes were analyzed from healthy fish and in CyHV-2 challenged fish. Based upon the real time PCR results the gene expression varied among the genes and in tissues. The expression levels of the housekeeping genes were then compared and evaluated with the RefFinder web tool which analyses results using four different algorithms - BestKeeper, delta Ct, geNorm and NormFinder. EF1AA was ranked to be the best gene in healthy fish by BestKeeper and geNorm analysis. The delta Ct and NormFinder algorithm have found 18S to be a stable gene in healthy fish but 18S was given to be least expressed in challenged fish. ACTB was also given as a stable gene by geNorm analysis in both healthy and challenged fish. Also, in CyHV-2 challenged fish, EF1AA was identified as the best gene by all the three analysis except by BestKeeper analysis, where it has ranked GADPH as the best housekeeping gene. Expression of the four candidate reference genes differed across all tissue types tested, inferring that a thorough study of the reference genes is necessary for cross tissue comparison. These results can be further used in the immune gene response study of goldfish infected with any viral pathogen to develop better health strategies in the disease management of goldfish aquaculture.

Clinicogenetic Profile, Treatment Modalities, and Mortality Predictors of Gaucher Disease: A 15-Year Retrospective Study.

INTRODUCTION: Gaucher disease (GD) is a rare autosomal recessive lysosomal storage disorder, in which biallelic pathogenic variants in the Glucosidase beta acid (GBA) gene result in defective functioning of glucosylceramidase that causes deposition of glucocerebroside in cells. GD has 3 major types namely, non-neuronopathic (type I), acute neuronopathic (type II), and chronic neuronopathic (type III). Definite treatment options are limited and expensive. They succumb early to the disease, if untreated. There is paucity of studies from the Indian subcontinent, which elicit the factors resulting in their premature mortality. MATERIALS AND METHODS: A retrospective study was carried out in a tertiary care setting of South India to assess the clinical profile, mutation spectrum, and various management strategies (only supportive therapy, enzyme replacement therapy [ERT], substrate reduction therapy [SRT] haematopoietic stem cell transplant [HSCT]), and mortality predictors of patients with GD from 2004 to 2019. A Kaplan-Meier survival curve was plotted. In silico predictions were performed for novel variants. RESULTS: There were 60 patients with all types of GD seen over the study period of 15 years. Their median age at diagnosis was 2 years. The median follow-up was for 5 years (interquartile range [IQR] = 2-8). The overall mortality rate was 35%; however, it was only 10% in those receiving definite treatment. Mortality was higher (47.5%) by more than 4 folds in those only on supportive therapy. The median survival from the time of diagnosis was 6.3 years (IQR = 3.5-10.8) in the definite treatment group and 3.5 years (IQR = 1-5) in those on supportive therapy. The Kaplan-Meier survival analysis showed significant (p value 0.001) mortality difference between these groups. The multiple logistic regression analysis found the neuronopathic type (OR = 5) and only supportive therapy (OR = 6.3) to be the independent risk factors for premature mortality. CONCLUSION: GD is a rare disease with a high mortality rate, if left untreated. ERT and SRT are the definitive treatments which increase the survival. In resource-limited settings like India, with higher prevalence of the neuronopathic type, HSCT may be a more suitable definitive treatment option, due to its one-time intervention and cost, assuming similar efficacy to ERT. However, the efficacy and safety of HSCT in GD needs to be established further by substantial patient numbers undergoing it.

Alazami syndrome: Report of three Indian patients with phenotypic spectrum from adolescence to adulthood.

Alazami syndrome (ALAZS) (MIM 615071) is a rare autosomal recessive disorder characterized by short stature, dysmorphic facial features, developmental delay, and impaired intellect. It was first reported in a Saudi Arabian family in 2012. Three Indian patients affected with ALAZS, one boy aged 13 years and other two sisters in their 40s are presented. These patients had few unreported dysmorphic facial features: high arched eyebrows and dental overcrowding. No microcephaly was noted in the sisters. One of the sisters did not have short stature. The boy also presented with unilateral buphthalmos of left eye. All three of them have been identified to harbor novel variants in LARP7.

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2).

BACKGROUND: Herpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line. METHODS: Caudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz's L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish. RESULTS: The cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30th, 40th and 56th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Transcription factor OsNF-YB9 regulates reproductive growth and development in rice.

MAIN CONCLUSION: OsNF-YB9 controls heading by affecting expression of regulators of flowering. It affects the development of the reproductive meristem by interacting with MADS1 and controlling expression of hormone-related genes. Nuclear Factor-Y (NF-Y) family of transcription factors takes part in many aspects of growth and development in eukaryotes. They have been classified into three subunit classes, namely, NF-YA, NF-YB and NF-YC. In plants, this transcription factor family is much diverged and takes part in several developmental processes and stress. We investigated NF-Y subunit genes of rice (Oryza sativa) and found OsNF-YB9 as the closest homologue of LEAFY COTYLEDON1. OsNF-YB9 delayed the heading date when ectopically expressed in rice. Expression of several heading date regulating genes such as Hd1, Ehd1, Hd3a and RFT1 were altered. OsNF-YB9 overexpression also resulted in morphological defects in the reproductive organs and led to pseudovivipary. OsNF-YB9 interacted with MADS1, a key regulator of floral development. This NF-Y subunit acted upstream to several transcription factors as well as signalling proteins involved in brassinosteroid and gibberellic acid metabolism and cell cycle. OsNF-YB9 and OsNF-YC12 interacted in planta and the latter also delayed heading in rice upon overexpression suggesting its involvement in a similar pathway. Our data provide new insights into the rice heading date pathway integrating these OsNF-Y subunit members to the network. These features can be exploited to improve vegetative growth and yield of rice plants in future.

Establishment and characterization of a caudal fin-derived cell line, AOF, from the Oscar, Astronotus ocellatus.

Astronotus ocellatus, commonly called the oscar, is one of the popular cichlids among aquarium hobby. The present study deals with the development and characterization of a new cell line from caudal fin of A. ocellatus. The cell line was cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28 °C. The optimum temperature and FBS concentration for cell growth were tested with temperature ranges from 20 to 37 °C and FBS concentrations of 5-20% at 28 °C. The Astronotus ocellatus fin cell line has been subcultured 45 times since its development and the modal chromosome number (2n) is 48. The cell line is composed mainly of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies. The cell line was cryopreserved at different passage levels and the revival efficiency showed 80% survival rate. Partial sequence amplification and sequencing of two genes, mitochondrial 16S ribosomal RNA and cytochrome oxidase I, confirmed the origin of cell line. The cell line did not show Mycoplasma contamination. The cells showed good transfection efficiency when transfected with 2 µg of pAcGFP1-N1 expression vector. The extracellular products of fish bacterial pathogens viz., Aeromonas hydrophila and A. caviae, were cytotoxic to AOF cells but were not susceptible to Cyprinid herpes virus 2. The development of AOF cell line will have significant applications in fish virology and will prove useful to isolate pathogens in the event of sudden viral disease outbreak and for the development of vaccines and diagnostic kits.

Ceruloplasmin, a moonlighting protein in fish.

Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The current knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin is highly conserved throughout the vertebrate evolution. Cupredoxin, a multi-cupper blue protein is believed to be the evolutionary precursor of ceruloplasmin with three trinuclear and three mononuclear copper binding sites. There are 20 copper-binding residues in ceruloplasmin gene out of which 16 residues are conserved in fish. This ceruloplasmin gene is being characterized in zebrafish (Danio rerio), rohu (Labeo rohita), Indian medaka (Oryzias melastigama), catfish (Ictalurus punctatus), icefish (Chionodraco rastrospinosus), goldfish (Carassius auratus) and yellow perch (Perca flaviscens). The complete coding sequence of fish ceruloplasmin gene is around 3.2 kb which codes for 1000 to 1100 amino acid residues. The size of ceruloplasmin gene sequence in fish ranges around 13 kb containing 20 exons and 19 introns. Liver is the major site of synthesis in fish. Increased expression of this gene during bacterial infection in channel catfish and rohu suggested its potential involvement in bacterial disease response in fish. It has been found to serve as an indirect marker for selection against Aeromonas hydrophila resistance in rohu carp. Ceruloplasmin expression is also evident during parasitic infection in few fish species. The role of this gene is well studied during inflammatory response to hormonal, drug and heavy metal mediated toxicity in fish. Overall, ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions.

An Efficient Strategy Combining SSR Markers- and Advanced QTL-seq-driven QTL Mapping Unravels Candidate Genes Regulating Grain Weight in Rice.

Development and use of genome-wide informative simple sequence repeat (SSR) markers and novel integrated genomic strategies are vital to drive genomics-assisted breeding applications and for efficient dissection of quantitative trait loci (QTLs) underlying complex traits in rice. The present study developed 6244 genome-wide informative SSR markers exhibiting in silico fragment length polymorphism based on repeat-unit variations among genomic sequences of 11 indica, japonica, aus, and wild rice accessions. These markers were mapped on diverse coding and non-coding sequence components of known cloned/candidate genes annotated from 12 chromosomes and revealed a much higher amplification (97%) and polymorphic potential (88%) along with wider genetic/functional diversity level (16-74% with a mean 53%) especially among accessions belonging to indica cultivar group, suggesting their utility in large-scale genomics-assisted breeding applications in rice. A high-density 3791 SSR markers-anchored genetic linkage map (IR 64 × Sonasal) spanning 2060 cM total map-length with an average inter-marker distance of 0.54 cM was generated. This reference genetic map identified six major genomic regions harboring robust QTLs (31% combined phenotypic variation explained with a 5.7-8.7 LOD) governing grain weight on six rice chromosomes. One strong grain weight major QTL region (OsqGW5.1) was narrowed-down by integrating traditional QTL mapping with high-resolution QTL region-specific integrated SSR and single nucleotide polymorphism markers-based QTL-seq analysis and differential expression profiling. This led us to delineate two natural allelic variants in two known cis-regulatory elements (RAV1AAT and CARGCW8GAT) of glycosyl hydrolase and serine carboxypeptidase genes exhibiting pronounced seed-specific differential regulation in low (Sonasal) and high (IR 64) grain weight mapping parental accessions. Our genome-wide SSR marker resource (polymorphic within/between diverse cultivar groups) and integrated genomic strategy can efficiently scan functionally relevant potential molecular tags (markers, candidate genes and alleles) regulating complex agronomic traits (grain weight) and expedite marker-assisted genetic enhancement in rice.

Three Rice NAC Transcription Factors Heteromerize and Are Associated with Seed Size.

NACs are plant-specific transcription factors (TFs) involved in multiple aspects of development and stress. In rice, three NAC TF encoding genes, namely ONAC020, ONAC026, and ONAC023 express specifically during seed development, at extremely high levels. They exhibit significantly strong association with seed size/weight with the sequence variations located in the upstream regulatory region. Concomitantly, their expression pattern/levels during seed development vary amongst different accessions with variation in seed size. The alterations in the promoter sequences of the three genes, amongst the five rice accessions, correlate with the expression levels to a certain extent only. In terms of transcriptional properties, the three NAC TFs can activate and/or suppress downstream genes, though to different extents. Only ONAC026 is localized to the nucleus while ONAC020 and ONAC023 are targeted to the ER and cytoplasm, respectively. Interestingly, these two proteins interact with ONAC026 and the dimers localize in the nucleus. Trans-splicing between ONAC020 and ONAC026 results in three additional forms of ONAC020. The transcriptional properties including activation, repression, subcellular localization and heterodimerization of trans-spliced forms of ONAC020 and ONAC026 are different, indicating toward their role as competitors. The analysis presented in this paper helps to conclude that the three NAC genes, which are associated with seed size, have independent as well as overlapping roles during the process and can be exploited as potential targets for crop improvement.

Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice.

We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17-79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9-21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, "Oryza ISM-ILP marker" database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice.

Epidemiological study of canine parvovirus infection in and around Bhubaneswar, Odisha, India.

AIM: An epidemiological study of canine parvovirus infection in dogs in and around Bhubaneswar, Odisha was conducted between December 2012 to March 2013 and prevalence rate was studied on the basis of age, breed, and sex. MATERIALS AND METHODS: A total of 71 fecal samples from suspected diarrheic dogs were collected in sterile phosphate buffer saline (10% W/V) and examined by polymerase chain reaction (PCR) for detection of canine parvo virus infection, followed by epidemiological study in relation to age, breed, and sex. RESULTS: Of 71 samples analyzed, 29 (40.85%) were found to be positive by PCR assay. The infection was higher in Deshi/local breeds (34.48%), followed by German shepherd (17.24%), equal incidences in mixed and Labrador retriever (10.34%), Rottweiler and German spitz showed 6.90% each and finally lower incidences in four breeds (3.45%) such as Dalmatians, Nea politan mastiff, Pug and Great Dane. Age-wise prevalence study revealed the infection being more in the age group of 3-6 months (41.37%), followed by equal incidences of 27.59% in 1-3 months and 6-12 months age group, and a low incidence in age groups above 12 months (3.45%). The incidence was predominantly higher in males (86.21%) than females (13.79%). CONCLUSIONS: The epidemiological analysis revealed that the breed wise prevalence was found to be more in Deshi breeds as compared to others, age groups below 6 months were found to be more prone to parvovirus infection and males were mostly infected.