Browsing by Author "Ruchi Singh"

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Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress
(Taylor and Francis Inc., 2015) Manoj K. Mishra; Gaurav Singh; Shalini Tiwari; Ruchi Singh; Nishi Kumari; Pratibha Misra
Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at-1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both b-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. © 2015 Taylor & Francis Group, LLC.
Comparative Study of the Signal-to-Noise Ratio and Gain in Photorefractive Materials
(Springer Science and Business Media, LLC, 2013) Ruchi Singh; R.A. Yadav; D.P. Singh
We calculate the dependence of the signal-to-noise ratio and gain on the angle α between the pump and signal beams and the beam ratio β in Cu-doped potassium-sodium-strontium-barium niobate (Cu:KNSBN) crystal and compare them with the experimental results available in the literature. We find good qualitative agreement between the calculated and experimental results. Also we study variations of SNR and gain with α and β in BaTiO3 crystal. Experimental data for this crystal are not available. © 2013 Springer Science+Business Media New York.
Dependence of space charge field and gain coefficient on the applied electric field in photorefractive materials
(2011) Ruchi Singh; M.K. Maurya; T.K. Yadav; D.P. Singh; R.A. Yadav
Intensity dependent space charge field and gain coefficient in the photorefractive medium due to the two interfering beams have been calculated by solving the material rate equations in presence of externally applied dc electric field. The gain coefficient has been studied with respect to variations in the input intensity, modulation depth, concentration ratio and normalized diffusion field in the absence and presence of the externally applied dc electric field. Space charge field has also been computed by varying the intensity ratio in the presence and absence of the externally applied dc electric field. It has been found that the rate of change of the space charge field with the normalized dc field decreases with the increasing intensity ratio for different values of the normalized diffusion field. It has also been found that the externally applied dc electric field has appreciable effect only when it is larger than the diffusion field. © 2010 Elsevier Ltd. All rights reserved.
Effect of applied electric field and grating spacing on performance of ring resonator
(Institute of Physics Publishing, 2015) Ruchi Singh; R.A. Yadav; D.P. Singh
In this paper we analyze the effect of grating spacing on relative oscillation intensity and two-beam coupling gain of a ring resonator in presence of an applied electric field. The photorefractive crystal used in the ring resonator is KNSBN. It is found that the relative oscillation intensity increases exponentially with grating spacing in presence as well as in absence of the applied electric field, and approaches to a saturation state after some grating spacing. The increase is however slower under applied field condition, hence, the saturation state is reached at higher value of the grating spacing. The two-beam coupling gain decreases with grating spacing for both conditions on electric field. The decrease is however less abrupt in presence of applied electric field. It is found that the saturation state occurs for the grating spacing over 2 to 4 μm. © Published under licence by IOP Publishing Ltd.
Effect of applied electric field on coupling gain constant in Sn2P2S6 crystal
(Optical Society of America (OSA), 2014) Ruchi Singh; R.A. Yadav; D.P. Singh
Coupling gain constant as a function of applied electric field and grating spatial frequency at small modulation depth for Sn2P2S6 crystal is analysed theoretically. The computed results are compared with the experimental results and are found to be reasonably good. © OSA 2014.
Effect of applied electric field on coupling gain constant in Sn2P2S6 crystal
(Optical Society of America (OSA), 2014) Ruchi Singh; R.A. Yadav; D.P. Singh
Coupling gain constant as a function of applied electric field and grating spatial frequency at small modulation depth for Sn2P2S6 crystal is analysed theoretically. The computed results are compared with the experimental results and are found to be reasonably good. © OSA 2014.
Effect of Grating Spacing on Response Time and Sensitivity in Photorefractive Materials at Large Modulation Depth
(2013) Ruchi Singh; D.P. Singh; R.A. Yadav
Photorefractive response time and photorefractive sensitivity are two important parameters characterizing photorefractive materials. We study theoretically the effect of the grating spacing on these parameters at large modulation depth in the absence and presence of an applied electric field for some of the most promising photorefractive materials, namely, LiNbO3, KNSBN, SPS, BGO, and GaAs. We find that the response time increases with increasing grating spacing for LiNbO3 and KNSBN, whereas the response time decreases with increasing grating spacing for SPS, BGO, and GaAs. The photorefractive sensitivity is mainly affected by the mobility-lifetime product in the presence of an applied electric field. © 2013 Springer Science+Business Media New York.
Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress
(Public Library of Science, 2017) Deepak Kumar Deep; Ruchi Singh; Vasundhra Bhandari; Aditya Verma; Vanila Sharma; Saima Wajid; Shyam Sundar; V. Ramesh; Jean Claude Dujardin; Poonam Salotra
Background: Miltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani. Methodology: L. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance. Results: LdRelapse parasites exhibited higher IC50both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites. Conclusion: The present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL. © 2017 Deep et al.
Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance, Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants
(2013) Manoj K. Mishra; Pankaj Chaturvedi; Ruchi Singh; Gaurav Singh; Lokendra K. Sharma; Vibha Pandey; Nishi Kumari; Pratibha Misra
Background:Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant's adaptation to abiotic stress.Methodology:The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.Results:The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.Conclusions:Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases. © 2013 Mishra et al.
Sterol glycosyltransferases required for adaptation of Withania somnifera at high temperature
(Blackwell Publishing Ltd, 2017) Gaurav Singh; Manish Tiwari; Surendra P. Singh; Ruchi Singh; Surendra Singh; Pramod A. Shirke; Prabodh K. Trivedi; Pratibha Misra
Heat is a major environmental stress factor that confines growth, productivity, and metabolism of plants. Plants respond to such unfavorable conditions through changes in their physiological, biochemical and developmental processes. Withania somnifera, an important medicinal plant, grows in hot and dry conditions, however, molecular mechanisms related to such adaptive properties are not known. Here, we elucidated that members of the sterol glycosyltransferases (SGT) gene family play important roles in the survival of W. somnifera under adverse conditions through maintaining the integrity of the membrane. SGTs are enzymes involved in sterol modifications and participate in metabolic flexibility during stress. Silencing of WsSGT members, for instance WsSGTL1, WsSGTL2 and WsSGTL4, was inimical for important physiological parameters, such as electron transport rate, photochemical quantum yield, acceptor side limitation, non-photochemical quenching (NPQ), Fv/Fm and net photosynthetic rate, whereas stomatal conductance, transpiration rate and dark respiration rates (Rds) were increased. Decreased NPQ and increased Rds helped to generate significant amount of ROS in the Wsamisgt lines. After heat stress, H2O2, lipid peroxidation and nitric oxide production increased in the Wsamisgt lines due to high ROS generation. The expression of HSPs in Wsamisgt lines might be involved in regulation of physiological processes during stress. We have also observed increased proline accumulation which might be involved in restricting water loss in the Wsamisgt lines. Taken together, our observations revealed that SGTL enzyme activity is required to maintain the internal damages of the cell against high temperature by maintaining the sterol vs sterol glycosides ratio in the membranes of W. somnifera. © 2017 Scandinavian Plant Physiology Society
Study of oscillation performance characteristics of unidirectional photorefractive ring resonators: Cavity detuning dependence
(American Institute of Physics Inc., 2015) Ruchi Singh; R.A. Yadav; D.P. Singh
Single unidirectional photorefractive ring resonator (SUPRR) and coupled unidirectional photorefractive ring resonator (CUPRR) were considered with BaTiO3 and LiNbO3 crystals as inter-cavity photorefractive media. The relative oscillation intensity and oscillation frequency shift of two SUPRRs with BaTiO3 and LiNbO3 crystals in their cavities, and four CUPRRs with BaTiO3-BaTiO3, LiNbO3-LiNbO3, BaTiO3-LiNbO3 and LiNbO3-BaTiO3 combinations were analysed relative to cavity detuning parameters and compared the oscillation performance of SUPRR with that of CUPRR. In SUPRR, maximum value of relative oscillation intensity is found for BaTiO3 as compared to that of LiNbO3 for zero cavity detuning. In CUPRR, the oscillation intensity in secondary cavity increases with increasing cavities detuning whereas in primary cavity it decreases with increasing cavities detuning. Major finding of this study is that the BaTiO3-LiNbO3 combination of the coupled system provides the highest relative oscillation intensity. © 2015 Author(s).
Theoretical study of properties of BaTiO3 material with grating spacing and light intensity
(2013) Ruchi Singh; D.P. Singh; R.A. Yadav
The effects of the grating spacing, light intensity, concentration ratio, wavelength and applied electric field on coupling gain constant of two light beams entering in BaTiO3 material have been theoretically studied. It is found that the coupling gain constant decreases with increasing light intensity; each of them reaches a maximum value at some specific grating spacing. The photoconductivity and phase shift, variations in BaTiO3 have also been studied and these are found to be similar to those in reduced KNbO3 material. © 2012 Elsevier GmbH. All rights reserved.
Theoretical study of SNR in photorefractive materials
(2013) Ruchi Singh; D.P. Singh; R.A. Yadav
Dependence of signal to noise ratio with angle θ, formed by the bisector of the angle between pump and signal beams with the crystal c-axis has been theoretically studied and compared with the experimental results The agreement between the theory and experiment is found to be good only qualitatively. © 2013 AIP Publishing LLC.
Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani
(Elsevier Ltd, 2017) Aditya Verma; Vasundhra Bhandari; Deepak Kumar Deep; Shyam Sundar; Jean Claude Dujardin; Ruchi Singh; Poonam Salotra
Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glycosomal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania. © 2017
Utility of Blood as the Clinical Specimen for the Molecular Diagnosis of Post-Kala-Azar Dermal Leishmaniasis
(American Society for Microbiology, 2021) Keerti Kaumudee Dixit; V. Ramesh; Shreya Upadhyay; Abhishek Kumar Singh; Om Prakash Singh; Shyam Sundar; Ruchi Singh; Poonam Salotra
The countries in the Indian subcontinent have reported a dramatic decline in visceral leishmaniasis (VL) cases. However, the presence of the parasite reservoir in the form of post-kala-azar dermal leishmaniasis (PKDL), a dermal sequel of VL, is a hurdle in attaining VL elimination. Presently employed clinical specimens for the diagnosis of PKDL include skin biopsy specimens and slit skin smears. In this study, the use of blood as a clinical specimen was investigated in different manifestations of PKDL in India. This is a bicentric study (National Institute of Pathology, Indian Council of Medical Research [ICMR], New Delhi, and Institute of Medical Sciences [IMS], Banaras Hindu University, Varanasi), with 215 participants (120 PKDL patients and 95 controls). Highly sensitive quantitative real-time PCR (Q-PCR) and field-deployable loop-mediated isothermal amplification (LAMP) were employed using blood samples for diagnosis. Promising sensitivities of 77.50% (95% confidence interval [CI], 69.24 to 84.05%) for Q-PCR and 70.83% (95% CI, 62.16 to 78.22%) for LAMP were obtained for the diagnosis of PKDL. Further, enhanced sensitivities of 83.33% (95% CI, 71.28 to 90.98%) and 77.78% (95% CI, 65.06 to 86.80%) for Q-PCR and LAMP, respectively, were recorded for the detection of macular cases. The study revealed an inverse correlation between the parasite load estimated in slit and blood samples, thereby favoring the use of blood for the diagnosis of the macular variant, which may be missed due to scant parasite loads in the slit. This study is the first to propose the promising potential of blood as a clinical specimen for accurate diagnosis of PKDL, which would aid in fast-tracking VL elimination. © 2021 American Society for Microbiology.
Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)
(Public Library of Science, 2018) Keerti Kaumudee Dixit; Sandeep Verma; Om Prakash Singh; Dharmendra Singh; Akhil Pratap Singh; Ratan Gupta; Narendra Singh Negi; Pradeep Das; Shyam Sundar; Ruchi Singh; Poonam Salotra
Background: The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL. Methodology: The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy. Principal findings: A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUC ROC ) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%). Conclusions/Significance: The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost. © 2018 Dixit et al. http://creativecommons.org/licenses/by/4.0/.