Insect cell culture in research: Indian scenario

Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies

Ganjam virus

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratoryacquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs

Background & objectives: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Methods: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID50) and indirect immunofluorescence assay (IFA). Results: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Interpretation & conclusions: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics

Biochemical Effects of Exercise on a Fasciocutaneous Flap in a Rat Model.

Importance: An overwhelming amount of data suggest that cardiovascular exercise has a positive effect on the mind and body, although the precise mechanism is not always clear. Objective: To assess the clinical and biochemical effects of voluntary cardiovascular exercise on pedicled flaps in a rodent model. Design, Setting, and Participants: Eighteen adult Sprague-Dawley male rats were randomized into a resting animal group (RAG) (n=9) and an exercise animal group (EAG) (n=9) for 14 days (July 23, 2013, through July 30, 2013). A pedicled transposition flap was performed on the ventral surface of the rat, and biopsy specimens were taken from the proximal, middle, and distal portions on postoperative days 0, 2, 5, and 9. Flap survival was analyzed planimetrically, and biopsy specimens were analyzed by hematoxylin-eosin-stained microscopy and immunoblotting. The housing, exercise, surgery, and analysis of the rats were conducted at a single basic science research laboratory at the tertiary care center campus of Thomas Jefferson University in Philadelphia, Pennsylvania. Exposures: The rats were caged for 14 days or housed in a cage connected to an exercise wheel and pedometer. Main Outcomes and Measures: Study measures were gross and micrographic necrosis and expression of proteins within cell survival and apoptosis pathways. Results: A total of 18 rats were studied, 9 in the RAG and 9 in the EAG. the mean (SEM) amount of necrosis in flaps was 41.3% (3%) in the RAG rats and 10.5% (3.5%) in the EAG rats (P \u3c .001). Immunoblotting revealed increased Caspase-9 activity resulting in poly-(adenosine diphosphate-ribose) polymerase 1 cleavage in the RAG vs the EAG, as well as lower phosphorylated protein kinase B (also known as Akt), signal transducer and activator of transcription 3, and total B-cell leukemia/lymphoma 2 protein levels. Throughout the postoperative period, the cumulative vascular endothelial growth factor A levels of the EAG flaps were significantly higher than those of the RAG flaps (2.30 vs 1.25 fold induction [FI], P = .002), with differences of 2.76 vs 1.54 FI in the proximal segment, 2.40 vs 1.20 FI in the middle segment, and 1.90 vs 0.79 FI in the distal segment. A similar response was noted when comparing phosphorylated Akt, with cumulative mean (SEM) p-Akt expression levels of 0.62 (0.04) for RAG and 1.98 (0.09) for EAG (P = .002 between the 2 groups). Conclusions and Relevance: Voluntary preoperative exercise improves survival in pedicled fasciocutaneous flaps; the EAG rats had less necrosis, decreased apoptotic markers, and increased amounts of vascular endothelial growth factor A and prosurvival proteins. These results have implications to increase flap survival in other mammal populations, such as humans. Level of Evidence: 3

Superconducting Material Diagnostics using a Scanning Near-Field Microwave Microscope

We have developed scanning near-field microwave microscopes which can image electrodynamic properties of superconducting materials on length scales down to about 2 $\mu$m. The microscopes are capable of quantitative imaging of sheet resistance of thin films, and surface topography. We demonstrate the utility of the microscopes through images of the sheet resistance of a YBa2Cu3O7-d thin film wafer, images of bulk Nb surfaces, and spatially resolved measurements of Tc of a YBa2Cu3O7-d thin film. We also discuss some of the limitations of the microscope and conclude with a summary of its present capabilities.Comment: 6 pages with 9 figures, Proceedings of the Applied Superconductivity Conference 199

Testing a new analytic model for gravitational lensing probabilities

We study gravitational lensing with a multiple lens plane approach, proposing a simple analytical model for the probability distribution function (PDF) of the dark matter convergence, kappa, for the different lens planes in a given cosmology as a function of redshift and smoothing angle, theta. The model is fixed solely by the variance of kappa, which in turn is fixed by the amplitude of the power spectrum, sigma_8. We test the PDF against a high resolution Tree-Particle-Mesh simulation and find that it is far superior to the Gaussian or the lognormal, especially for small values of theta << 1 arcmin and at large values of kappa relevant to strong lensing. With this model, we predict the probabilities of strong lensing by a single plane or by multiple planes. We find that for theta ~ 10 arcsec, a single plane accounts for almost all (~ 98%) of the strong lensing cases for source redshift unity. However, for a more typical source redshift of 4, about 12% of the strong lensing cases will result from the contribution of a secondary clump of matter along the line of sight, introducing a systematic error in the determination of the surface density of clusters, typically overestimating it by about 2-5%. We also find that matter inhomogenieties introduce a dispersion in the value of the angular diameter distance about its cosmological mean. The probable error relative to the mean increases with redshift to a value of about 8% for z ~ 6 and theta ~ 10 arcsec.Comment: Accepted for publication in ApJ, 13 pages, 12 figures, revised version, references added, section 6 expande

A task and performance analysis of endoscopic submucosal dissection (ESD) surgery

BACKGROUND: ESD is an endoscopic technique for en bloc resection of gastrointestinal lesions. ESD is a widely-used in Japan and throughout Asia, but not as prevalent in Europe or the US. The procedure is technically challenging and has higher adverse events (bleeding, perforation) compared to endoscopic mucosal resection. Inadequate training platforms and lack of established training curricula have restricted its wide acceptance in the US. Thus, we aim to develop a Virtual Endoluminal Surgery Simulator (VESS) for objective ESD training and assessment. In this work, we performed task and performance analysis of ESD surgeries. METHODS: We performed a detailed colorectal ESD task analysis and identified the critical ESD steps for lesion identification, marking, injection, circumferential cutting, dissection, intraprocedural complication management, and post-procedure examination. We constructed a hierarchical task tree that elaborates the order of tasks in these steps. Furthermore, we developed quantitative ESD performance metrics. We measured task times and scores of 16 ESD surgeries performed by four different endoscopic surgeons. RESULTS: The average time of the marking, injection, and circumferential cutting phases are 203.4 (σ: 205.46), 83.5 (σ: 49.92), 908.4 s. (σ: 584.53), respectively. Cutting the submucosal layer takes most of the time of overall ESD procedure time with an average of 1394.7 s (σ: 908.43). We also performed correlation analysis (Pearson's test) among the performance scores of the tasks. There is a moderate positive correlation (R = 0.528, p = 0.0355) between marking scores and total scores, a strong positive correlation (R = 0.7879, p = 0.0003) between circumferential cutting and submucosal dissection and total scores. Similarly, we noted a strong positive correlation (R = 0.7095, p = 0.0021) between circumferential cutting and submucosal dissection and marking scores. CONCLUSIONS: We elaborated ESD tasks and developed quantitative performance metrics used in analysis of actual surgery performance. These ESD metrics will be used in future validation studies of our VESS simulator

Genetic divergence of Chikungunya viruses in India (1963-2006) with special reference to the 2005-2006 explosive epidemic

Re-emergence of Chikungunya (CHIK), caused by CHIK virus, was recorded in India during 2005-2006 after a gap of 32 years, causing 1.3 million cases in 13 states. Several islands of the Indian Ocean reported similar outbreaks in the same period. These outbreaks were attributed to the African genotype of CHIK virus. To examine relatedness of the Indian isolates (IND-06) with Reunion Island isolates (RU), full-genome sequences of five CHIK virus isolates representative of different Indian states were determined. In addition, an isolate obtained from mosquitoes in the year 2000 (Yawat-2000), identified as being of the African genotype, and two older strains isolated in 1963 and 1973 (of the Asian genotype), were sequenced. The IND-06 isolates shared 99.9 % nucleotide identity with RU isolates, confirming involvement of the same strain in these outbreaks. The IND-06 isolates shared 98.2 % identity with the Yawat-2000 isolate. Of two crucial substitutions reported for RU isolates in the E1 region, M269V was noted in the Yawat-2000 and IND-06 isolates, whereas D284E was seen only in the IND-06 isolates. The A226V shift observed with the progression of the epidemic in Reunion Island, probably associated with adaptation to the mosquito vector, was absent in all of the Indian isolates. Three unique substitutions were noted in the IND-06 isolates: two (T128K and T376M) in the Nsp1 region and one (P23S) in the capsid protein. The two Asian strains showed 99.4 % nucleotide identity to each other, indicating relative stability of the virus. No evidence of recombination of the Asian and African genotypes, or of positive selection was observed. The results may help in understanding the association, if any, of the unique mutations with the explosive nature of the CHIK outbreak

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10²-10¹⁰(r²= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10⁰PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10²PFU/ml). Vero and PS cell-lines (1.2 × 10³PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p

Cosmological Parameters from Pre-Planck CMB Measurements

Recent data from the WMAP, ACT and SPT experiments provide precise measurements of the cosmic microwave background temperature power spectrum over a wide range of angular scales. The combination of these observations is well fit by the standard, spatially flat LCDM cosmological model, constraining six free parameters to within a few percent. The scalar spectral index, n_s = 0.9690 +/- 0.0089, is less than unity at the 3.6 sigma level, consistent with simple models of inflation. The damping tail of the power spectrum at high resolution, combined with the amplitude of gravitational lensing measured by ACT and SPT, constrains the effective number of relativistic species to be N_eff = 3.28 +/- 0.40, in agreement with the standard model's three species of light neutrinos.Comment: 5 pages, 4 figure