Technical validation of a new microfluidic device for enrichment of CTCs from large volumes of blood by using buffy coats to mimic diagnostic leukapheresis products.

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.

Complete Genome Sequence of Brucella abortus SKN 13 Isolated from Placenta of Aborted Cattle in Gujarat, India.

Brucella abortus is generally known to cause brucellosis in cattle and buffalo. Here, we report the draft genome sequence of Brucella abortus SKN 13, isolated from aborted cattle placenta in the area of Gujarat, India, providing precious resources for comparative genomic analyses of Brucella field strains.

Pressure ulcer management in paraplegic patients with a novel negative pressure device: a randomised controlled trial.

OBJECTIVE: A randomised controlled trial to compare negative pressure wound therapy (NPWT) using our innovative negative pressure device (NPD) and the standard pressure ulcer (PU) wound dressing of in traumatic paraplegia patients. METHOD: This study was conducted in the Department of Orthopaedic Surgery at King George's Medical University, Lucknow, India. Traumatic paraplegia patients with sacral pressure ulcers of stage 3 and 4 were randomised into two groups, receiving either standard wound dressings or NPWT with NPD. The outcomes monitored were length, width (surface area), depth of PU, exudates, discharge, tissue type (necrotic, slough and red granulating tissue), and cost-effectiveness during 0 to 9 weeks follow-up. RESULTS: Length and width were significantly (p<0.01) decreased in NPWT group as compared with standard care group at week 9. At weeks 1, 2 and 3, depth was significantly (p<0.05) higher in NPWT group, whereas at week 9 a significant reduction (p=0.01) was observed. Exudates were significantly (p=0.001) lower in NPWT group at weeks 4 and 9. Conversion of slough into red granulation tissue was significantly higher in NPWT group (p=0.001). Discharge became significantly (p=0.001) lower in NPWT at week 2 and no discharge was observed after week 6. In all parameters, decrease was larger in NPWT group compared with standard care, which was significant for exudates type (p=0.03) and tissue type (p=0.004). CONCLUSION: Our NPD is better than standard wound care procedures and cost-effective for management of PU.

Comparison of molecular and microscopic technique for detection of Theileria annulata from the field cases of cattle.

AIM: Tropical theileriosis is fatal hemoprotozoal disease of dairy animals caused by Theileria annulata. The aim of the present study was to detect the T. annulata and comparison of results of molecular and microscopic techniques. MATERIALS AND METHODS: A total of 52 blood samples were collected from the cattle suspected for theileriosis across the Banaskantha district. All the samples were screened for theileriosis using Giemsa's staining technique and polymerase chain reaction (PCR). RESULTS: Total of 17 (32.69%) and 24 (46.15%) samples were found positive for theileriosis by microscopic examination and PCR test, respectively. It revealed that the study area is endemic for theileriosis, and the microscopic technique has 70.83% sensitivity and 100% specificity with respect to PCR technique. CONCLUSION: It may be concluded from the present study that the PCR is comparatively sensitive technique than microscopic examination and may be recommended to use in the field for screening of theileriosis in the study area, where a high prevalence of diseases have been reported due to intensive dairy farming.

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses.

During 1960-80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences. All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A-D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782-1934) and DENV-4 (1719-1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938-1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India. The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.

Development of real time PCR for detection and quantitation of Dengue Viruses.

BACKGROUND: Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study. RESULTS: The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples. CONCLUSION: We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.

Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas.

Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas at different concentrations (0.5, 1, 1.5, 3 and 5 mgl(-1)) for 10 min were studied. A cocktail of three strains of each targeted organism (100 microl) was spotted onto the surface of the strawberries (approximately 8-9 log ml(-1)) separately followed by air drying, and then treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity. Approximately a 4.3-4.7 logCFU reduction per strawberry of all examined bacteria was achieved by treatment with 5 mgl(-1) ClO(2) for 10 min. The inactivation kinetics of E. coli O157:H7, L. monocytogenes and S. enterica were determined using first-order kinetic models to establish D-values and z-values. The D-values of E. coli, L. monocytogenes and S. enterica were 2.6+/-0.2, 2.3+/-0.2 and 2.7+/-0.7 min, respectively, at 5 mgl(-1) ClO(2). The z-values of E. coli, L. monocytogenes and S. enterica were 16.8+/-3.5, 15.8+/-3.5 and 23.3+/-3.3 mgl(-1), respectively. Furthermore, treatment with ClO(2) gas significantly (p < or = 0.05) reduced the initial microflora (mesophilic, psychrotrophic bacteria, yeasts and molds) on strawberries. Treatment with ClO(2) gas did not affect the color of strawberries and extended the shelf-life to 16 days compared to 8 days for the untreated control.

Synthesis and bronchodilator activity of new quinazolin derivative.

Taking lead from a naturally occurring quinazolin vasicine, a number of compounds were developed and evaluated for bronchodilator and anti-allergic activities. One of these compounds was 2,4-diethoxy-6,7,8,9,10,12-hexahydroazepino[2,1-b]quinazolin-12-one, hereinafter named 95-4, exhibited marked bronchodilator activity evaluated on contracted trachea or constricted tracheo-bronchial tree. On intestinal smooth muscle too it showed relaxant effect. Tracheal relaxant effect was not found to be mediated through beta-adrenoceptors. Cumulative dose-response study with acetylcholine and histamine indicated for its non-specific direct effect on smooth muscles. 95-4 was found to be more potent than theophylline and less to that of salbutamol on dose basis. Tested by a number of experimental models, it was found devoid of anti-allergic activity. It was also found to be free from any adverse effect. 95-4 due to its marked bronchial muscle relaxant effect can find use in conditions associated with spasm of bronchial muscles.

Bilateral ptosis following wasp sting.

We report a case of bilateral ptosis due to wasp sting which was completely treatable.

Cannabis psychosis and acute schizophrenia. a case-control study from India.

Twenty cases of cannabis psychosis were compared with a control group of 20 patients with 'acute schizophrenic episode' on a number of demographic, clinical, illness-related and outcome variables in a case-control study design using a retrospective chart review. The two groups were comparable on demographic, past and family histories of mental illness, premorbid personality, psychomotor activity, Schneiderian first-rank symptoms and mild cognitive deficits. The cases, in contrast to the control group, had a psychosis of shorter duration characterized by reactive and congruent affect, relative absence of schizophrenic formal thought disorder and a predominantly polymorphic clinical picture. Relapse was always preceded by cannabis use. This study suggests that, in spite of certain overlaps, 'cannabis psychosis' may still be considered nosologically distinct from schizophrenia in India. The implication of the study is that the role of cannabis in any acute psychosis should be investigated carefully so as to prevent an overdiagnosis of schizophrenia.

Determination of carbidic carbon in steels.

An attempt has been made to develop a simple analytical technique that can readily be used in an industrial laboratory to isolate and quantify the precipitated carbides in steel samples. Attempt has also been made to correlate this value with the thermodynamically predicted values. There is good agreement between the predicted and experimental results. More work is in progress to develop this technique for the preparation of Certifed Reference Materials with dissolved and precipitated carbon in steels.

A community day care programme for psychiatric patients: the role of occupational therapy.

This paper examines the need for and role of occupational therapy in the organization and functioning of a community day care centre for psychiatric patients in a developing country. It highlights the debilitating effects of institutionalization associated with a previously established system of institutional care and explores the role of occupational therapy in facilitating the move away from that approach towards community-based day care centres for such patients. The paper describes the community care programme; the patients attending the programme; the methods used by occupational therapists to evaluate patients; and the effectiveness of the various individual, group and family focused methods of treatment deployed.

Autoradiographic localization of 3H-digoxin binding by neural cells in the medulla.

The purpose of this investigation was to localize binding sites for the cardiac glycoside digoxin in the medulla of the rat in vivo. Adult male Sprague-Dawley rats were injected (IV) with 3H-digoxin and killed 30 minutes later. Autoradiographs of medullas showed evidence of 3H-digoxin binding to small- and medium-sized neural cells in the regions of the nucleus solitarius, dorsal motor nucleus of the vagus, area postrema, and in the zone between the area postrema and the underlying neuropil. However, the parasympathetic preganglionic neurons of the dorsal motor nucleus were not labeled. The 3H-digoxin-labeled cells in the medulla were located mainly in the commissural and medial portions of nucleus solitarius at the level of the area postrema. Animals injected with unlabeled digoxin followed by 3H-digoxin showed reduced binding of radioactivity. The small- and medium-sized neurons of the caudal portions of the nucleus solitarius are internuncial in position with respect to cardiovascular afferents of the glossopharyngeal and vagus nerves and sympathetic and parasympathetic cardiovascular efferent neurons of the medulla. The results of this study suggest that these 3H-digoxin-labeled cells, presumably neurons of nucleus solitarius, may possess high affinity binding sites for digoxin. Further, the area postrema, which lacks a blood-brain barrier, may provide a portal of entry for 3H-digoxin into regions of the medulla known to contain neurons that play a role in the regulation of cardiac rhythm.