Development of Diagnostic and Vaccine Markers Through Cloning, Expression, and Regulation of Putative Virulence-Protein-Encoding Genes of Aeromonas hydrophila

Vijai Singh,Dharmendra Kumar Chaudhary,Indra Mani,Rohan Jain,B.N. Mishra 한국미생물학회 2013 The journal of microbiology Vol.51 No.3

Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of diseases in fish, amphibians, reptiles, and humans. In fish it causes several disease symptoms including tail and skin rot, and haemorrhagic septicemia; in human it causes soft-tissue wound infection and diarrhoea. The pathogenesis of A. hydrophila is multifactorial, but the mechanism is unknown so far. It is considered to be mediated by expression and secretion of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. A number of the putative virulence-protein-encoding genes that are present in the genome of A. hydrophila have been targeted by PCR for molecular diagnosis. These significant genes are also targeted for over-production of proteins by cloning and expression methods. In this review, we emphasize recent progress in the cloning, expression, and regulation of putative virulence-protein-encoding genes of A. hydrophila for a better understanding of the pathogenesis and also help to provide effective strategies for control of diseases.

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

Vijai Singh Karwasara,Vinod Kumar Dixit 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3

The effect of culture medium nutrients ongrowth and alkaloid production by plant cell cultures ofNothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae)was studied with a view to increasing the productionof the alkaloid camptothecin, a key therapeutic drug usedfor its anticancer properties. Amongst the various sugarstested with Murashige and Skoog (MS) medium, such asglucose, fructose, maltose, and sucrose, maximum accumulationof camptothecin was observed with sucrose. Highnitrate in the media supports the biomass, while highammonium enhances the camptothecin content. Selectivefeeding of 60 mM total nitrogen with a NH4?/NO3- balanceof 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over thecontrol culture (28.5 lg/g DW). Furthermore, the sucrosefeeding strategy greatly stimulated cell biomass and camptothecinproduction. A modified MS medium was developedin the present study, which contained 0.5 mMphosphate, a nitrogen source feeding ratio of 50/10 mMNH4?/NO3- and 3 % sucrose with additional 2 % sucrosefeeding (added on day 12 of the cell culture cycle) with10.74 lM naphthaleneacetic acid and 0.93 lM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higherintracellular and extracellular camptothecin content overthe control culture (29.2 and 8.2 lg/g DW), respectively.

A simple, rapid and sensitive spectrofluorimetric method for the determination of camptothecin

Karwasara, Vijai Singh,Nahata, Alok,Dixit, Vinod Kumar 경희한의학연구센터 2012 Oriental Pharmacy and Experimental Medicine Vol.12 No.2

Camptothecin (CPT) represents a clinically useful class of anticancer agent. Proper identification and quantitation of the CPT in the plant extracts and in-vitro cell culture extracts is fundamental to assess the CPT content and its biosynthetic potential in plants. A simple, sensitive and rapid, spectrofluorimetric method has been developed and validated for the quantitative estimation of camptothecin. The method was validated in terms of linearity (2-20 ng/ml), precision (intra-day variation below 0.15, interday variation below 1.2), and accuracy (98.0 to 100.2%). The limit of detection and limit of quantification for CPT were found to be 0.10 ng/ml and 0.36 ng/ml, respectively. The developed spectrofluorimetric method provides a rapid and cost effective method for the routine analysis of CPT in plant extracts and tissue culture samples. The developed method was successfully used for the estimation of CPT in natural plant extracts and cell culture extracts. The Nothapodytes nimmoniana callus cells having nearly 3-fold higher CPT content over the leaf (0.005%) explant of the plant. The highest CPT content was found in the stem part (0.092%) followed by the fruit (0.088%). The method is simple, sensitive and precise; it can be used for the routine quality control testing of formulations containing CPT.

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

Karwasara, Vijai Singh,Dixit, Vinod Kumar 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a $NH_4{^+}/NO_3{^-}$ balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture ($28.5{\mu}g/g$ DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM $NH_4{^+}/NO_3{^-}$ and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with $10.74{\mu}M$ naphthaleneacetic acid and $0.93{\mu}M$ kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and $8.2{\mu}g/g$ DW), respectively.

Pregnancy - associated human listeriosis: Virulence and genotypic analysis of Listeria monocytogenes from clinical samples

Dharmendra Kumar Soni,Durg Vijai Singh,Suresh Kumar Dubey 한국미생물학회 2015 The journal of microbiology Vol.53 No.9

cccListeria monocytogenes, a life-threatening pathogen, poses severe risk during pregnancy, may cause abortion, fetal death or neonatal morbidity in terms of septicemia and meningitis. The present study aimed at characterizing L. monocytogenes isolated from pregnant women based on serotyping, antibiotic susceptibility, virulence genes, in vivo pathogenicity test and ERIC- and REP-PCR fingerprint analyses. The results revealed that out of 3700 human clinical samples, a total of 30 (0.81%) isolates [12 (0.80%) from placental bit (1500), 18 (0.81%) from vaginal swab (2200)] were positive for L. monocytogenes. All the isolates belonged to serogroup 4b, and were + ve for virulence genes tested i.e. inlA, inlC, inlJ, plcA, prfA, actA, hlyA, and iap. Based on the mice inoculation tests, 20 isolates showed 100% and 4 isolates 60% relative virulence while 6 isolates were non-pathogenic. Moreover, 2 and 10 isolates were resistant to ciprofloxacin and cefoxitin, respectively, while the rest susceptible to other antibiotics used in this study. ERIC- and REP-PCR collectively depicted that the isolates from placental bit and vaginal swab had distinct PCR fingerprints except a few isolates with identical patterns. This study demonstrates prevalence of pathogenic strains mostly resistant to cefoxitin and/or ciprofloxacin. The results indicate the importance of isolating and characterizing the pathogen from human clinical samples as the pre-requisite for accurate epidemiological investigations. Listeria monocytogenes, a life-threatening pathogen, poses severe risk during pregnancy, may cause abortion, fetal death or neonatal morbidity in terms of septicemia and meningitis. The present study aimed at characterizing L. monocytogenes isolated from pregnant women based on serotyping, antibiotic susceptibility, virulence genes, in vivo pathogenicity test and ERIC- and REP-PCR fingerprint analyses. The results revealed that out of 3700 human clinical samples, a total of 30 (0.81%) isolates [12 (0.80%) from placental bit (1500), 18 (0.81%) from vaginal swab (2200)] were positive for L. monocytogenes. All the isolates belonged to serogroup 4b, and were + ve for virulence genes tested i.e. inlA, inlC, inlJ, plcA, prfA, actA, hlyA, and iap. Based on the mice inoculation tests, 20 isolates showed 100% and 4 isolates 60% relative virulence while 6 isolates were non-pathogenic. Moreover, 2 and 10 isolates were resistant to ciprofloxacin and cefoxitin, respectively, while the rest susceptible to other antibiotics used in this study. ERIC- and REP-PCR collectively depicted that the isolates from placental bit and vaginal swab had distinct PCR fingerprints except a few isolates with identical patterns. This study demonstrates prevalence of pathogenic strains mostly resistant to cefoxitin and/or ciprofloxacin. The results indicate the importance of isolating and characterizing the pathogen from human clinical samples as the pre-requisite for accurate epidemiological investigations.

Molecular Detection and Genotyping of Fusarium oxysporum f. sp. psidii Isolates from Different Agro-Ecological Regions of India

Rupesh Kumar Mishra,Brajesh Kumar Pandey,Vijai Singh,Amita John Mathew,Neelam Pathak,Mohammad Zeeshan 한국미생물학회 2013 The journal of microbiology Vol.51 No.4

Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop),causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings,where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer,with the amplified DNA fragments ranging from 200–2,000bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However,genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.

Vaginal Dose, Toxicity and Sexual Outcomes in Patients of Cervical Cancer Undergoing Image Based Brachytherapy

Rai, Bhavana,Dhanireddy, Bhaswanth,Patel, Firuza Darius,Kumari, Reena,Oinam, Arun Singh,Simha, Vijai,Sharma, Suresh Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.8

Background: The aim of the study was to evaluate the vaginal dose and toxicity in patients of cervical cancer treated with image guided brachytherapy at our institute. Materials and Methods: Thirty-five patients treated with image based brachytherapy for cervical cancer were included. Vaginal contouring was done on MRI at brachytherapy and with CT scans of subsequent brachytherapy fractions. Dose volume parameters (DVH) were reported in accordance with the GEC-ESTRO guidelines. These were correlated with vaginal toxicity (assessed by CTCAE version 3) and quality of sexual life assessed at one year of completion of treatment. Results: Vaginal shortness was observed in 22 out of 30 (62.8%) patients, Nine (25.7%) had vaginal dryness and in 10 (28.5%) patients, there was contact bleeding. No association could be demonstrated between the dose volume parameters and vaginal toxicity in the present study. Conclusions: The lack of association between dose volume parameters of vagina with vaginal morbidity may be due to uncertainties involved in the delineation of vaginal wall and dosimetry. Future research is required to accurately define vaginal dose distribution to study its correlation with vaginal morbidity. Vaginal morbidity needs to be documented in order to improve the sexual outcome in these patients.