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Biomedical subjects

P K Dash

Publications and source records attributed to P K Dash.

At least 19 recordsLinked to original sources
PubMed2006

Rapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay.

The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 x 10(8) to 2 x 10(2) copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 x 10(8) to 2 x 10(1) copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63 degrees C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

Acute Disease↗
PubMed2006

Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus.

The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the clinical samples was 10(2) to 10(5) PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.

Antibodies, Viral↗
PubMed2006

Comparison of a dipstick dot-ELISA with commercial assays for anti-dengue virus IgM antibodies.

Dengue virus infections have undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Dengue virus causes life-threatening complications characterized by dengue hemorrhagic fever and dengue shock syndrome. No standard validated test systems are available for serological diagnosis of dengue virus infection. This creates problems in the diagnosis and proper management of patients. Evaluation of a Defense Research and Development Establishment (DRDE) dengue virus dipstick dot-ELISA test, developed in house, vis-à-vis commercially available immunodiagnostic kits was carried out for detection of IgM antibodies. The DRDE dengue dipstick dot-ELISA was performed on the basis of the dot-ELISA principle. Commercial tests, namely, the Panbio Dengue IgM Capture ELISA system (Panbio, Sinnamon Park, Australia) and Pathozyme Dengue M (Omega Diagnostics, Alva, UK), were performed according to the manufacturers' protocols. Ninety-one serum samples collected from the states of Kerala and Delhi, India during August and November of 2004 were used in the present study. The overall agreement among all three tests was found to be only 72.16% for IgM antibodies. Correlations between the DRDE dipstick dot-ELISA and the Panbio kit, between the DRDE dipstick dot-ELISA and the Pathozyme Dengue M kit, and between the Panbio kit and the Pathozyme Dengue M kit were found to be 96, 93, and 94%, respectively. Although the Panbio kit is widely used in various laboratories in India, the DRDE dipstick dot-ELISA promises to be a useful kit because of its field applicability and comparable sensitivity.

Animals↗
PubMed2005

Mechanism of ricin-induced apoptosis in human cervical cancer cells.

The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.

Apoptosis↗
PubMed2004

Immunotherapeutic potential of Ocimum sanctum (L) in bovine subclinical mastitis.

Immunotherapeutic potential of aqueous extract of Ocimum sanctum (O. sanctum) leaf in bovine sub-clinical mastitis (SCM) was investigated. Somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), phagocytic activity and Phagocytic index and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase content were evaluated after intramammary infusion of aqueous leaf extract of O. sanctum. The results revealed that the aqueous extract of O. sanctum treatment reduced the TBC and increased neutrophil and lymphocyte counts with enhanced phagocytic activity and phagocytic index. Similarly, the lysosomal enzymes contents of the milk polymorphonuclear cells (PMNs) were also enhanced significantly in animals treated with the extract. The results suggest that the crude aqueous extract of O. sanctum (leaf) possesses some biologically active principles that are antibacterial and immunomodulatory in nature. As such, the present wok substantiates the therapeutic use of medicinal herb and also emphasizes on the potential of the commonly available non-toxic substances to enhance the mammary immunity.

Acid Phosphatase↗
PubMed2004

Performance in long-term memory tasks is augmented by a phosphorylated growth factor receptor fragment.

To elucidate the role of enhanced phosphoinositide-3-kinase (PI3-kinase) activity in memory, a synthetic phosphopeptide (TAT-YPMDM) containing the p85 regulatory subunit receptor-binding motif (YXXM) coupled to the cell transduction domain of HIV-TAT protein was employed. This phosphopeptide bound the p85 subunit of PI3-kinase, and was internalized by both granule and pyramidal neurons when injected into the hippocampus. Increased lipid kinase activity and enhanced phosphorylation of the PI3-kinase substrates Akt (protein kinase B) and ribosomal S6 kinase were associated with TAT-YPMDM administration. Bilateral infusion of the phosphopeptide into the dorsal hippocampus after training improved performance in three hippocampus-dependent memory tasks: contextual fear conditioning, trace fear conditioning, and the Morris water maze. Both the biochemical and behavioral effects of the TAT-YPMDM phosphopeptide could be blocked by wortmannin. No effect was observed when a nonphosphorylated peptide (TAT-YMDM), or a second, unrelated phosphopeptide (TAT-YPLDL) was utilized. In addition, infusion of the TAT-YPMDM phosphopeptide did not interfere with memory acquisition or 4 hr memory. In addition, pretesting administration did not affect the ability to recall a previously established long-term memory. These findings suggest that stimulation of PI3-kinase activity by phosphorylated receptor fragments containing the YMDM motif augments long-term memory.

Amino Acid Motifs↗
PubMed2004

Effect of age on atrial contribution to ventricular filling after balloon mitral valvuloplasty in mitral stenosis.

OBJECTIVE: Ventricular filling takes place during the conduit and pump functions of the atrium. While studying whether relief of mitral valve obstruction improves atrial filling, the effect of age on atrial contribution to ventricular filling was studied before and after balloon mitral valvuloplasty (BMV) and on follow-up at 1 year. METHODS: Patients with mitral stenosis (MS) and sinus rhythm (n = 59) were divided into group I (< 18 years, n = 13), group II (< 30 years, n = 29) and group III (> 30 years, n = 17). Two-dimensional mitral valve area (MVA in cm2), transmitral mean gradient (MG in mm Hg), velocity time integral (VTI in cm) of mitral valve flow, VTI contributed by atrial systole (A-VTI), difference between total VTI and A-VTI (E-VTI), percentage contribution of A-VTI to the total VTI (A-%) and difference between A-% before and after BMV (delta-A-%) were noted. Follow-up data was obtained at 1 year. The change in A-% at follow-up (A-%-FU) was calculated as the difference between A-% before BMV and A-% at follow-up. RESULTS: There was a similar increase in MVA with a reduction in MG among the three groups. Among the three groups, total VTI and E-VTI before and after BMV were similar. Before BMV, in all the groups, A-VTI and A-% were similar. After BMV, there was increase in A-VTI and A-% in all the groups with a trend to be more in younger patients. A-VTI was significantly higher in group I only. But E-VTI had decreased significantly in all groups and tended to be less in younger patients. In younger patients, delta-A-% after BMV was significantly higher (13.2 +/- 7.6, 7.9 +/- 5.1 and 6.5 +/- 4.5, respectively, in groups I, II and III; p < 0.01). Correlation coefficient of age against delta-A-% was -0.55 (p < 0.01). Correlation coefficients of delta-A-% against post-BMV-MVA and MG were not good. At follow-up of 11.3+/-1.2 months, changes achieved in total VTI, A-VTI, E-VTI and A-% were maintained. Total VTI, A-VTI, E-VTI and A-% were similar at the time of follow-up on comparing the three groups. But younger patients had significantly higher A-%-FU (12.1 +/- 5.8, 9.4 +/- 4.6 and 7.3 +/- 3.1, respectively, in groups I, II and III; p < 0.01). CONCLUSIONS: Prior to BMV, there is an age related reduction in atrial contribution to ventricular filling that improves with relief of MS. Advancing age reduces the immediate and late recovery of atrial contribution after BMV. This may be due to increasing left atrial fibrosis with age that prevents an improvement in atrial pump function. The differential improvement in atrial function in younger patients warrants earlier intervention in MS to achieve better recovery of atrial function.

Adolescent↗
PubMed2004

The activity of milk leukocytes in response to a water-soluble fraction of Mycobacterium phlei in bovine subclinical mastitis.

The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2003

Expression of cytochromes P450 4F4 and 4F5 in infection and injury models of inflammation.

Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.

Animals↗
PubMed2003

Comparative analysis of mRNA levels in the frontal cortex and the hippocampus in the basal state and in response to experimental brain injury.

Damage to the frontal cortex and to the hippocampus, both in terms of cell loss and neuronal dysfunction, is thought to underlie many of the neurological and behavioural consequences of traumatic brain injury (TBI). Several studies have indicated that the hippocampus is particularly susceptible to central nervous system insults, whereas the frontal cortex possesses relatively higher capacities for regeneration and plasticity. It has been postulated that dissimilarities in the gene expression profiles in these structures, both in the normal and the postinjury states, may underlie these differences. In order to explore this issue, mRNA samples taken from the frontal cortex and the hippocampus of uninjured animals were subjected to high-density microarray analysis. The analysis indicated that the mRNA levels of 65 genes were differentially expressed between these two brain regions. Among these, genes involved in intracellular signalling, neurotransmitter release, and genes encoding for channels and receptors were identified. Samples taken from animals injured using controlled cortical impact (a model of TBI) showed altered mRNA levels for 341 frontal cortex genes 24 h following injury. These genes can be broadly classified into one of 12 functional classes: cell cycle, metabolism, reactive oxygen metabolism, inflammation, receptors, channels and transporters, signal transduction, cytoskeleton, membrane proteins, neuropeptides, growth factors, and proteins involved in transcription/translation. The expression profile of these genes is compared to the expression profile of 241 genes in the hippocampus 24 h following cortical impact injury as previously reported by our laboratory. In addition to genes previously reported in the literature, this study found several genes that have not been associated with TBI. The functional implications of changes in the expression of some of these genes are discussed.

Animals↗
PubMed2003

Prediction of atrial fibrillation in patients with severe mitral stenosis--role of atrial contribution to ventricular filling.

OBJECTIVE: Atrial contribution to ventricular filling was studied to assess its role in predicting the future development of atrial fibrillation (AF) in patients with severe mitral stenosis (MS) and sinus rhythm. DESIGN: Two hundred and eight patients with severe MS and sinus rhythm were followed up for 1 year. Baseline data were compared between group I (who developed AF at follow-up) and group II (who maintained sinus rhythm). Left atrial size, severity of MS, velocity time integral (VTI) of mitral valve flow and VTI due to atrial systole (A-VTI) were noted. Percentage contribution of A-VTI to the total VTI (A-%) was calculated. Sensitivity and specificity of A-% to predict the onset of AF was obtained. RESULTS: Left atrial size, severity of MS and total VTI were similar in the two groups. Group I patients were older (31.1 +/- 9.1 and 18.4 +/- 6.5 years, respectively, p < 0.03) with smaller A-VTI (5.3 +/- 2.2 and 6.7 +/- 3.4 cm, respectively, p < 0.01) and A-% (8.9 +/- 1.8 and 11.2 +/- 2.7, respectively, p < 0.003). A-% of <9% (mean value of A-VTI in group I) had high sensitivity (84%, positive predictive value 76%) and specificity (80%, negative predictive value 87%) to predict the development of AF. CONCLUSION: Atrial contribution to ventricular filling is reduced in patients prone to develop AF (due to inefficient left atrial contraction, much before its dilatation). It can be used for early identification of patients likely to develop AF with high sensitivity and specificity. It is simple, easily available, cost-effective and will guide earlier intervention and more frequent follow-up. There is a preclinical loss in atrial pump function much before the eventual onset of AF.

Adolescent↗
PubMed2002

The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal death and alters the plasticity (e.g. morphology) of surviving neurons. Both of these events contribute to TBI-associated neurological deficits, such as memory dysfunction. Although a majority of current research is directed towards identifying biochemical cascades responsible for cell death, little is known about mechanisms of altered neuronal plasticity following TBI. Extracellular signal-regulated kinases (Erk1 and 2) play a critical role in growth and have been implicated in long-lasting neuronal plasticity and memory storage. The activation of Erk following TBI was investigated utilizing an antibody that specifically binds to dually phosphorylated Erk. Using this antibody, we report that lateral cortical impact injury in rats increases Erk phosphorylation both in the cortex and the hippocampus as early as 10 min post-injury. Double immunostaining experiments using either a neuron-specific or an astroglial-specific marker show that the active Erk is localized almost exclusively in neuronal cells. Furthermore, the increase in phospho-Erk immunoreactivity was initially localized to axons and at later time points was observed to be predominantly in the cell soma. This suggests that Erk redistributed over time and may play a role in retrograde signaling. Administration of inhibitors of the Erk cascade worsened retrograde amnesia, impaired performances in hippocampus- and amygdala-dependent memory tasks, and exacerbated motor deficits following TBI. Furthermore, inhibition of this cascade did not have any overt effects on cell survival, but altered neuronal morphology as detected by a dendritic-specific marker. These findings suggest that the Erk cascade plays an essential role for the maintenance of neuronal function and plasticity following TBI.

Animals↗
PubMed2002

Serological & virological investigation of an outbreak of dengue fever in Gwalior, India.

BACKGROUND & OBJECTIVES: An outbreak of febrile illness occurred between September to November 2001 in Gwalior, Madhya Pradesh affecting individuals mostly in the age group < 30 yr. A total of 312 febrile indoor patients suspected to have dengue infection were investigated. METHODS: The investigation included examination of blood samples from patients for dengue specific IgM and IgG antibodies, isolation of virus in suckling mouse pups and in C(6/36) cell line followed by confirmation and typing through reverse transcriptase-PCR and nested PCR. RESULTS: The serological analysis of the 312 samples indicated 65 per cent positivity of which 21 per cent are of recent infection as indicated by the presence of IgM antibody and 78 per cent are found to be secondary in nature by showing the presence of IgG and/or IgM antibodies. The RT-PCR analysis of patients' sera employing dengue virus group specific conserved amplimer confirmed the etiological agent as dengue complex by showing the characteristic 511 bp amplicons. None of the antibody positive samples were found to be positive by RT-PCR. A total of 13 (6%) samples positive by RT-PCR, were processed for virus isolation in mouse pups and in C(6/36) cells. Of these 9 samples (80%) were confirmed positive for virus isolation as identified by RT-PCR. INTERPRETATION & CONCLUSION: The typing of isolates by nested PCR employing serotype specific amplimer revealed 119 bp amplicon characteristic of dengue virus type-2 and thus confirming the outbreak attributed to dengue virus type-2.

Adult↗
PubMed2001

Enhanced neurogenesis in the rodent hippocampus following traumatic brain injury.

Recent studies have shown that neurogenesis in the dentate gyrus of the rodent hippocampus continues throughout life. Several physiological and pathological conditions have been reported to alter the rate of progenitor cell division resulting in the increased production of mature granule neurons. Excitotoxic and mechanical lesions of the granule cell layer also stimulate the proliferation of precursor cells suggesting that the death of pre-existing granule neurons may act as a trigger for enhanced neurogenesis. Hippocampal pyramidal neurons, and to a lesser extent granule neurons, have been reported to die as a result of traumatic brain injury in rodents. To determine if the proliferation of precursor cells is enhanced as a result of brain injury in rodents, newly divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). Traumatic brain injury increased the production of BrdU-labeled cells in the dentate gyrus with a maximal rate observed at 3 days post-injury. These cells, a proportion of which co-localize with the immature neuronal marker TOAD-64, implanted themselves into the granule cell layer where they accumulated over time. When examined 1 month post-injury, the majority of BrdU-labeled cells co-labeled with the mature neuronal marker calbindin. These findings show that traumatic brain injury increases neurogenesis in the granule cell layer and suggests that these new cells may contribute to the function of the hippocampus.

Animals↗
PubMed2001

Enhanced lipid peroxide levels in the erythrocytes of calves with haemoglobinuria.

Eight 6-9 month old calves, showing clinical signs of intermittent haemoglobinuria, even after treatment with an antipiroplasmal drug (4,4-diamidinodiazaminobenzene diaceturate), were examined for oxidative damage to their erythrocytes and the presence of hemoprotozoa in blood smears. Four calves without signs of haemoglobinuria served as controls. The blood smears from three of the eight calves contained piroplasms for Theileria annulata. Irrespective of the presence of piroplasms in their blood smears, the calves with haemoglobinuria had significantly (p < 0.01) lower haemoglobin concentrations (Hb) and packed cell volumes (PCV). The lipid peroxide level in the erythrocytes, but not in the plasma, of calves with red urine was significantly (p <0.05) higher than that for the controls. It is concluded that haemoglobinuria, irrespective of the presence of piroplasms in blood smears, is associated with oxidative stress to erythrocytes and peroxidation of polyunsaturated fatty acids of cell membrane.

Animals↗