Indranil Dasgupta

Virus-induced gene silencing: a versatile tool for discovery of gene functions in plants.

Virus-induced gene silencing (VIGS) is a technology that exploits an antiviral defense mechanism in plants as a tool for plant reverse genetics. VIGS circumvents the need for plant transformation, is methodologically simple and yields rapid results. Various VIGS vectors have been developed and have helped to unravel the functions of genes involved in processes such as disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.

Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium-mediated transfer

In nature, rice tungro disease is caused by an RNA and a DNA virus complex, but we have obtained an independently infectious clone of rice tungro bacilliform virus (RTBV) DNA. Infectivity could be demonstrated only when a more than unit-length copy was cloned in the Agrobacterium binary vector Bin 19 and agroinoculated into rice plants. Rice plants thus agroinfected with cloned RTBV DNA showed typical symptoms of tungro disease, presence of viral DNA and bacilliform particles, and could be used as a source of virus to infect healthy plants by the green leafhopper (Nephotettix virescens). The importance of this infectious clone in understanding the molecular biology of RTBV and the rice tungro disease is discussed.

Overweight, obesity and chronic kidney disease.

The sharp rise in the prevalence of chronic kidney disease (CKD) that parallels an increase in the prevalence of obesity in the recent years is a cause for great concern. CKD increases the rate of cardiovascular disease (CVD), development of end-stage renal disease, and leads to premature death. Although no direct causality link between obesity and CKD can yet be established, this appears highly likely. CKD should be regarded as a major complication of overweight and obesity, regardless of whether the association was independent or through the influence of diabetes, hypertension, CVD, metabolic syndrome and high fructose intake. We review the literature on the complex but positive association between obesity and CKD, the pathological effect of excess adiposity in kidney injury and the potential role of weight reduction therapy in reducing the burden of CKD.

A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors

Rice production is known to be severely affected by virus transmitting rice pests, brown planthopper (BPH) and green leafhopper (GLH) of the order hemiptera, feeding by phloem abstraction. ASAL, a novel lectin from leaves of garlic (Allium sativum) was previously demonstrated to be toxic towards hemipteran pests when administered in artificial diet as well as in ASAL expressing transgenic plants. In this report ASAL was targeted under the control of phloem-specific Agrobacterium rolC and rice sucrose synthase-1 (RSs1) promoters at the insect feeding site into popular rice cultivar, susceptible to hemipteran pests. PCR, Southern blot and C-PRINS analyses of transgenic plants have confirmed stable T-DNA integration and the transgenes were co-segregated among self-fertilized progenies. The T0 and T1 plants, harbouring single copy of intact T-DNA expression cassette, exhibit stable expression of ASAL in northern and western blot analyses. ELISA showed that the level of expressed ASAL was as high as 1.01% of total soluble protein. Immunohistofluorescence localization of ASAL depicted the expected expression patterns regulated by each promoter type. In-planta bioassay studies revealed that transgenic ASAL adversely affect survival, growth and population of BPH and GLH. GLH resistant T1 plants were further evaluated for the incidence of tungro disease, caused by co-infection of GLH vectored Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), which appeared to be dramatically reduced. The result presented here is the first report of such GLH mediated resistance to infection by RTBV/RTSV in ASAL expressing transgenic rice plant.

Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP

Summary.The biodiversity of geminiviruses associated with the Cassava Mosaic Disease (CMD) in India was investigated using PCR to specifically amplify the DNA of Indian cassava mosaic virus (ICMV) or Sri Lankan cassava mosaic virus (SLCMV) and also by using PCR to amplify specific viral genes, followed by digestion with different restriction endonucleases to obtain polymorphic patterns (PCR-RFLP). Results showed that both ICMV and SLCMV were present in mosaic-affected cassava; ICMV was geographically restricted to certain regions, whereas SLCMV was widespread. PCR-RFLP analysis showed that, in addition to ICMV-type and SLCMV-type patterns, a high proportion (40%) of the samples displayed novel patterns, some of which were localized in certain areas, whereas others were widely distributed.

Variation in the genome of rice tungro bacilliform virus: molecular characterization of six isolates

The DNA genomes of isolates of rice tungro bacilliform virus from Bangladesh, India, Indonesia, Malaysia and Thailand were cloned and compared with that of the type isolate from the Philippines. Restriction endonuclease maps revealed differences between the isolates and cross-hybridization showed that they fell into two groups, those from the Indian subcontinent and those from south-east Asian countries. The genomes of isolates from the Indian subcontinent contained a deletion of 64 bp when compared with those from south-east Asia. The implications of this variation are discussed.

Defective interfering DNAs of plant viruses

Several plant DNA viruses produce significant quantities of deleted versions of their DNA in the infected plants, which is generally correlated with a slowing down of replication process of the viral DNA. These deleted versions of the viral DNA are called Defective-Interfering (DI) DNA because of their inhibitory effect on the helper virus. The sizes of the DI-DNAs for different plant viruses can vary from one tenth of the size of the viral genome to one half and are mostly encapsidated. Sequence analysis suggests that DI-DNAs are formed by deletion, duplication, inversion, rearrangement and sometimes by insertion of non-viral DNA sequences involving the viral genome and its satellites. The role of the host plant in the formation of DI-DNAs is also important as DI-DNAs are readily formed in experimental hosts rather than their natural hosts. Symptom modulation by DI-DNAs is believed to be by competition for essential viral and host factors, thereby reducing the levels of the helper virus. Evidence is also emerging of its role in activating PTGS in the plant against viral transcripts, which is also likely to contribute towards symptom amelioration. The possibility of DI-DNAs playing a role towards the integration of pieces of viral DNA into plant genomes also exists. Most importantly, DI-DNAs have the potential to act as a tool in developing novel control strategies against viruses of crop plants and acting as gene expression/silencing vectors. Referee: Professor Henryk (Hanokh) Czosnek, Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Joint UK societies' 2014 consensus statement on renal denervation for resistant hypertension.

Resistant hypertension continues to pose a major challenge to clinicians worldwide and has serious implications for patients who are at increased risk of cardiovascular morbidity and mortality with this diagnosis. Pharmacological therapy for resistant hypertension follows guidelines-based regimens although there is surprisingly scant evidence for beneficial outcomes using additional drug treatment after three antihypertensives have failed to achieve target blood pressure. Recently there has been considerable interest in the use of endoluminal renal denervation as an interventional technique to achieve renal nerve ablation and lower blood pressure. Although initial clinical trials of renal denervation in patients with resistant hypertension demonstrated encouraging office blood pressure reduction, a large randomised control trial (Symplicity HTN-3) with a sham-control limb, failed to meet its primary efficacy end point. The trial however was subject to a number of flaws which must be taken into consideration in interpreting the final results. Moreover a substantial body of evidence from non-randomised smaller trials does suggest that renal denervation may have an important role in the management of hypertension and other disease states characterised by overactivation of the sympathetic nervous system. The Joint UK Societies does not recommend the use of renal denervation for treatment of resistant hypertension in routine clinical practice but remains committed to supporting research activity in this field. A number of research strategies are identified and much that can be improved upon to ensure better design and conduct of future randomised studies.

Biodiversity and recombination of cassava-infecting begomoviruses from southern India

Summary.Cassava mosaic disease (CMD) is caused by various begomoviruses of the family Geminiviridae leading to considerable crop losses in Africa and Asia. Recombination between their genomic components has generated new pathotypes with enhanced virulence in Africa. Here, we report about a survey on the biodiversity of begomoviruses in cassava from southern India (Tamil Nadu and Kerala states) performed in 2001 and 2002. Viral DNA A components from stem cuttings were analysed using polymerase chain reaction and restriction fragment length polymorphism. Eight representative examples were completely sequenced. The majority of DNA sequences (7 of 8) obtained were more closely related to that of Sri Lankan cassava mosaic virus (SLCMV) than of Indian cassava mosaic virus (ICMV). Only one sequence collected in Kerala was related to ICMV. The diversity of the SLCMV-like sequences was rather low compared to the variability of African viruses associated with cassava mosaic disease. Based on DNA A sequence data, all of these isolates should be classified as variants of SLCMV or ICMV. Phylogenetic analysis revealed mosaic structures within the DNA sequences which may indicate footprints of recombination events between ancestors of SLCMV and ICMV.

Uraemic Pruritus: Relief of Itching by Gabapentin and Pregabalin

Background: Pruritus (skin irritation or itching) is common in patients with chronic kidney disease (CKD) stages 4 and 5. It is associated with disrupted sleep, reduced quality of life, depression and increased mortality. A video of a patient describing the symptoms is at vimeo.com/49458473. Methods: We used gabapentin or pregabalin in 71 consecutive patients, 82% male. 25 had CKD stage 4 or 5, median eGFR = 17, range 9-30; 40 were on haemodialysis; 6 on peritoneal dialysis. Median itch severity score out of 10 = 8, range 6-10; median duration of itching = 6 months, range 0.5-240. Serum calcium ≤2.60 mmol/l (≤10.4 mg/dl) in 87% patients, phosphate ≤1.8 mmol/l (≤5.6 mg/dl) in 75%. 63% had used antihistamines and not gained relief. Starting dose of gabapentin 100 mg after dialysis or daily. Patients intolerant of gabapentin were offered pregabalin, starting dose 25 mg after dialysis or daily. Results: Gabapentin relieved itching in 47 patients (66%). A video of a patient describing the effect is at vimeo.com/49455976. 26 patients (37%) suffered side effects from gabapentin. Of 21 patients who stopped gabapentin due to side effects, 16 started pregabalin. Pregabalin relieved itching in 13 patients (81%). In total, gabapentin or pregabalin relieved itching in 60 patients (85%), median follow-up 2 months (range 1-8 months). Median itch severity out of 10 reduced from 8 to 1. Conclusions: Gabapentin or pregabalin relieved itching in 85% of 71 consecutively treated CKD patients. Patients should be advised about side effects and the drug initiated at a low dose. Patients intolerant of gabapentin may tolerate pregabalin.