Evaluation of Febrile Thrombocytopenia Cases in a South Indian Tertiary Care Hospital.

Objective: This study is aimed at analyzing the clinical symptomatology and hematological evaluation with an emphasis on platelet indices in relation to predicting the outcome of the febrile thrombocytopenic patients admitted in Coimbatore medical college hospital.. Methods: This is a prospective study involving 100 adult patients who presented to our hospital with fever and thrombocytopenia (platelet <1,50,000). This study excluded patients with known causes of thrombocytopenia like ITP and patients on chemotherapy etc. Results: Out of 100 patients 34 were dengue positive, 66 were dengue negative. Dengue specific symptoms like myalgia and retro-orbital pain were present in 58.88% of dengue positive and 10.60% of dengue negative patients. Laboratory evaluation revealed sharp rise in hematocrit with fall in platelet count in both the groups more significant in dengue positive group. Bleeding manifestation and rashes were 29.4% and 26.4% in dengue positive, 12.12% and 7.57% in dengue negative group respectively. MPV was significantly lower in patients with bleeding manifestations irrespective of platelet count in both the groups. Mortality in our study was 2%. Conclusion: MPV is an independent predictor of bleeding manifestation and poor outcome. Dengue virus may suppress the bone marrow as evidenced by alteration in MPV in addition to other mechanisms of thrombocytopenia.

High salinity induced expression profiling of differentially expressed genes in shrimp (Penaeus monodon).

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to high (55 ppt) salinity conditions. One each of the forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, signal transduction, cellular process, structural and membrane proteins, stress and osmoregulation. Gene expression levels in response to high salinity conditions at 2 weeks post salinity stress for some of the differentially expressed genes (Na(+)/K(+)-ATPase α-subunit, glutathione peroxidase, intracellular fatty acid binding protein, elongation factor 2, 14-3-3 like protein, penaeidin, translationally controlled tumor protein, transglutaminase and serine proteinase inhibitor B3) identified from SSH cDNA libraries were analysed by real-time RT-PCR. The highest gene expression levels was observed for Na(+)/K(+)-ATPase α-subunit in gill tissues (15.23-folds) and antennal glands (12.01-folds) and intracellular fatty acid binding protein in gut tissues (14.05-folds) respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.

Molecular cloning and characterization of acyl-CoA binding protein (ACBP) gene from shrimp Penaeus monodon exposed to salinity stress.

Acyl-CoA binding protein (ACBP), a protein present ubiquitously in wide range of organisms play significant role in transport of acyl groups for macromolecular biosynthesis involved in various functional and regulatory processes. In crustaceans, ACBP has functional role in growth, reproduction and temperature tolerance. In the present study, two suppression subtractive hybridization (SSH) cDNA libraries were performed using gut tissues of shrimp Penaeus monodon exposed to low (3 ppt) and high (55 ppt) salinity stress conditions. SSH library resulted in identification of differentially expressed genes that belonged to various functional classes such as the nucleic acid regulation and replication, defence proteins, allergen protein, signal transduction pathways, apoptosis, energy and metabolism, cell cycle regulation and hypothetical proteins. ACBP was identified as one of the differentially expressed gene in both the SSH libraries of shrimp P. monodon subjected to low and high salinity stress. The full-length cDNA of P. monodon ACBP gene was isolated and the sequence revealed 273 bp open reading frame encoding 90 amino acids with molecular mass of 10 kDa and pI 6.8. The ORF showed presence of four phosphorylation sites, with absence of signal peptide sequence and glycosylation sites. The deduced amino acid sequence of ACBP exhibited high sequence identity (92%) with ACBP class of protein identified from Fenneropenaeus chinensis. Real time PCR analysis of shrimps subjected to 3 ppt salinity conditions after 2 weeks revealed an increase in expression of ACBP transcripts, in the gut (28.08-folds), gills (11.71-folds) and in the muscle tissues (1.70-folds). Whereas, shrimps exposed to 55 ppt salinity conditions after 2 weeks exhibited increased ACBP transcript levels in the gut (11.95-folds), gills (1.052-folds) and muscle tissues (7.35-folds). The significant increase in expression levels of ACBP in various tissues of shrimps suggests a functional role of this gene in salinity stress tolerance and adaptation.

Identification and expression analysis of differentially expressed genes from shrimp (Penaeus monodon) in response to low salinity stress.

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to low (3 ppt) salinity conditions. Forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, cell signaling, cellular process, cytoskeleton and membrane structure, stress and osmoregulation. Gene expression levels in response to low salinity conditions at 2 weeks post salinity stress of thirteen selected differentially expressed genes identified from SSH cDNA libraries (14-3-3 like protein, crust in, lysozyme, arginine kinase, Naþ/Kþ-ATPase a-subunit, intracellular fatty acid binding protein, cathepsin B, anti-lipopolysaccharide factor, ferritin, ubiquitin conjugating enzyme E2, calreticulin, innexin 2 and heat shock protein 21) were analyzed by RT-PCR. The highest gene expression levels were observed for Naþ/Kþ-ATPase a-subunit (34.28-folds) in gill tissues, intracellular fatty acid binding protein (13.30-folds) in gut tissues and innexin 2 (14.43-folds) in muscle tissues respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.

Identification, cloning and expression analysis of Catechol-O-methyltransferase (COMT) gene from shrimp, Penaeus monodon and its relevance to salinity stress.

O-methyltransferase (OMT), a protein present ubiquitously in wide range of organisms plays significant role in methylation of small macro molecules for various functional and regulatory purposes. In crustaceans, OMT has functional role in growth, reproduction, ovarian development and molting. In the present study, suppression subtractive hybridization (SSH) performed using gill tissues of low (3ppt) and high (55ppt) salinity stressed shrimp Penaeus monodon resulted in identification of differentially expressed genes involved in signal transduction pathways, metabolism, defense proteins, DNA repair and synthesis, apoptosis, cell cycle regulation along with unknown and hypothetical proteins. Catechol-O-methyltransferase (COMT) a type of OMT was identified by SSH as one of the differentially expressed genes of shrimp P. monodon subjected to low and high salinity stress. The full length cDNA of COMT was cloned from the gills of P. monodon which consisted an open reading frame of 666 bp, encoding 221 amino acids. The ORF revealed one each of N-glycosylation and O-glycosylation sites and nine phosphorylation sites. The deduced amino acid sequence of COMT exhibited high sequence identity (92%) with COMT class of protein from Fenneropenaeus chinensis. Real time PCR analysis of the shrimp samples exposed to low salinity conditions at 3ppt revealed significant increase in expression of COMT transcripts in the guts at 24 h, 48 h, 1 week and 2 weeks, gills at 24 h and in the muscle tissues at 48 h, with maximum expression of the COMT levels by 5 fold in guts (1 week), 1 fold in gills (24 h) and 1.5 fold in muscle (48 h) respectively. The increased expression level of COMT at different time intervals in different tissues suggests a possible role of this gene in salinity stress tolerance in shrimps under low salinity conditions.

Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon).

Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 µg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 µg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.