De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

De novo transcriptome sequencing facilitates genomic resource generation in Tinospora cordifolia.

Tinospora cordifolia is known for its medicinal properties owing to the presence of useful constituents such as terpenes, glycosides, steroids, alkaloids, and flavonoids belonging to secondary metabolism origin. However, there is little information available pertaining to critical genomic elements (ESTs, molecular markers) necessary for judicious exploitation of its germplasm. We employed 454 GS-FLX pyrosequencing of entire transcripts and altogether ∼25 K assembled transcripts or Expressed sequence tags (ESTs) were identified. As the interest in T. cordifolia is primarily due to its secondary metabolite constituents, the ESTs pertaining to terpenoids biosynthetic pathway were identified in the present study. Additionally, several ESTs were assigned to different transcription factor families. To validate our transcripts dataset, the novel EST-SSR markers were generated to assess the genetic diversity among germplasm of T. cordifolia. These EST-SSR markers were found to be polymorphic and the dendrogram based on dice similarity index revealed three distinct clustering of accessions. The present study demonstrates effectiveness in using both NEWBLER and MIRA sequence read assembler software for enriching transcript-dataset and thus enables better exploitation of EST resources for mining candidate genes and designing molecular markers.

Potential Chemoprevention of 7,12-Dimethylbenz[a]anthracene Induced Renal Carcinogenesis by Moringa oleifera Pods and Its Isolated Saponin.

Present investigation shows that hydroethanolic extract of Moringa oleifera (MOHE) and its isolated saponin (SM) attenuates DMBA induced renal carcinogenesis in mice. Isolation of SM was achieved by TLC and HPLC and characterization was done using IR and (1)H NMR. Animals were pre-treated with MOHE (200 and 400 mg/kg body weight; p.o), BHA as a standard (0.5 and 1 %) and SM (50 mg/kg body weight) for 21 days prior to the administration of single dose of DMBA (15 mg/kg body weight). Administration of DMBA significantly (p < 0.001) enhanced level of xenobiotic enzymes. It enhanced renal malondialdehyde, with reduction in renal glutathione content, antioxidant enzymes and glutathione-S-transferase. The status of renal aspartate transaminase, alanine transaminase, alkaline phosphatase and total protein content were also found to be decreased along with increase in total cholesterol in DMBA administered mice. Pretreatment with MOHE and SM significantly reversed the DMBA induced alterations in the tissue and effectively suppressed renal oxidative stress and toxicity.

Antihepatotoxic activity of Euphorbia neriifolia extract against N-nitrosodiethylamine-induced hepatocarcinogenesis in mice.

OBJECTIVE: To scrutinize the protective role of hydroethanolic extract of Euphorbia neriifolia leaves (HEEN) against N-nitrosodiethylamine (DENA)-induced hepatocarcinogenesis in male Swiss albino mice. METHODS: Experimental mice were pretreated with 150 and 400 mg/kg of HEEN, or 0.5% and 1% of butylated hydroxyanisole (BHA) as a standard for 14 d prior to the administration of a single dose of 50 mg/kg of DENA. Levels of xenobiotic metabolizing enzymes such as cytochrome (Cyt) P450 and Cyt b5, activities of reduced glutathione (GSH), glutathione-S-transferase (GST), aspartate amino transaminase (AST), alanine amino transaminase (ALT) and alkaline phosphatase (ALP), and total protein and cholesterol content in the liver tissues were measured to determine the hepatotoxicity caused by DENA. RESULTS: The levels of Cyt P450 and Cyt b5 were significantly increased, and GST and GSH were significantly depleted after DENA treatment (P<0.01). The activities of AST, ALT and ALP, and the total protein content were also significantly dropped off (P<0.01). The total cholesterol level was markedly increased by DENA as compared with the normal group (P<0.01). However, the pre-supplementation of HEEN showed a remarkable amelioration of these abnormalities, and the levels of the antioxidant enzymes in the liver were significantly restored, which exhibited the dose-dependent protective effect against DENA-induced hepatotoxicity. CONCLUSION: HEEN exerts its chemopreventive effects by alleviating the xenobiotic enzymes and enhancing the levels of antioxidants and biochemical assays in DENA-induced carcinogenesis by reducing the formation of free radicals.

Renoprotective effects of Moringa oleifera pods in 7,12-dimethylbenz [a] anthracene-exposed mice.

OBJECTIVE: To investigate the potential of hydroethanolic extract of Moringa oleifera (MOHE) against 7, 12-dimethylbenz [a] anthracene (DMBA)-induced toxicity in male Swiss albino mice. METHODS: Experimental mice were respectively pretreated with 200 and 400 mg/kg of MOHE, and 0.5% and 1% of butylated hydroxyanisole (BHA) for two weeks prior to the administration of 15 mg/kg of DMBA, respectively. Levels of xenobiotic metabolizing enzymes such as cytochrome (Cyt) P450 and Cyt b5, activities of reduced glutathione (GSH) and glutathione-S-transferase (GST) and renal aspartate amino transaminase (AST), alanine amino transaminase (ALT) and alkaline phosphatase (ALP), and content of protein and total cholesterol were measured to determine the nephrotoxicity caused by DMBA and to elucidate the ameliorating role of M. oleifera. RESULTS: Single oral administration of 15 mg/kg of DMBA resulted in significant increases in Cyt P450 and Cyt b5 (P<0.01). The toxic effect of DMBA was justified by the significant decreases in the activities of GSH and GST in renal tissues (P<0.05). The levels of renal AST, ALT and ALP and protein content which are indicative of renocellular damage were also found decreased along with significant increase in total cholesterol content in DMBA-treated mice (P<0.01). The DMBA-induced alterations in the tissues were significantly reversed after pretreatment with 200 and 400 mg/kg of MOHE orally for 14 d (P<0.01). CONCLUSION: The effects of MOHE in enhancing the levels of antioxidants and enhancing the levels of biochemical assays in DMBA-induced carcinogenesis are by reducing the formation of free radicals. This study rationalizes the ethnomedicinal use of M. oleifera for the protection against nephrotoxicity induced by chemical carcinogens.

Molecular Characterization of Tinospora cordifolia (Willd.) Miers Using Novel g-SSR Markers and Their Comparison with EST-SSR and SCoT Markers for Genetic Diversity Study.

In the present study, novel genomic-SSR (g-SSR) markers generated in our laboratory were used to characterize Tinospora cordifolia and related species. The g-SSR marker was also compared with EST-SSR and SCoT markers used earlier in our laboratory to assess the genetic diversity of T. cordifolia. A total of 26 accessions of T. cordifolia and 1 accession each of Tinospora rumphii and Tinospora sinensis were characterized using 65 novel g-SSR markers. A total of 125 alleles were detected with 49 polymorphic g-SSR markers. The number of alleles per locus varied from 1-4 with a mean value of 2.55 alleles per locus. Mean PIC, gene diversity, and heterozygosity were estimated to be 0.33, 0.41, and 0.65, respectively. The two species, namely T. rumphii and T. sinensis, showed cross-species transferability of g-SSRs developed in T. cordifolia. The success rate of cross-species transferability in T. rumphii was 95.3% and 93.8% in T. sinensis, proving the usefulness of this marker in genetic diversity studies of related species. The Tinospora accessions were also used for molecular characterization using SCoT and EST-SSR markers and compared for genetic diversity and cross-species transferability. The PIC, gene diversity, heterozygosity, and principal coordinate analysis showed that g-SSR is the better maker for a genetic diversity study of T. cordifolia. Additionally, high cross-species transferability of g-SSRs was found (95.3% and 93.8%) compared to EST-SSRs (68.8% and 67.7%) in T. rumphii and T. sinensis, respectively.

Applicability of MIKE 21 to assess temporal and spatial variation in water quality of an estuary under the impact of effluent from an industrial estate.

This study aims at analysing the impact of wastewater load of industrial units in Haldia on the water quality of the Hoogly estuary using numerical modelling techniques. Modelling was never attempted in this region because it was generally felt that simulating such a complex system would not be easy with limited data availability but MIKE 21, a hydrodynamic and water quality model, was used to simulate BOD and DO profiles in the study area and simulation provided reasonably good predictions. A scenario assessment was also carried by increasing the flows from various sources to understand the site-specific relationships between pollution sources and water quality conditions and also to calculate the assimilative capacity of Hoogly with respect to waste discharged from the industrial units at Haldia. The results suggested that water quality of the canal towards closed Oil Jetty was highly deteriorated due to stagnant pond condition and discharges from a refinery. But large part of the canal was found to be well drained under tidal influence as a consequence of which both DO and BOD conform to the prescribed water quality standards most of the times. The impact of industrial waste load on the Hoogly was found to be negligible and the radius of influence was limited to about 200-300 m across the estuary and about 1.6 km along the shore during ebb conditions. Assimilative capacity of the region was estimated to be 837 kg/d as against the present load of 121 kg/d. The study suggested that the estuary had good dilution capacity and intense tidal mixing helped in rapidly diluting the pollutants.

Development of novel g-SSR markers in guava (Psidium guajava L.) cv. Allahabad Safeda and their application in genetic diversity, population structure and cross species transferability studies.

Dearth of genomic resources particularly, microsatellite markers in nutritionally and commercially important fruit crop, guava necessitate the development of the novel genomic SSR markers through the library enrichment techniques. Three types of 3' -biotinylated oligonucleotide probes [(CT)14, (GT)12, and (AAC)8] were used to develop microsatellite enriched libraries. A total of 153 transformed colonies were screened of which 111 positive colonies were subjected for Sanger sequencing. The clones having more than five motif repeats were selected for primer designing and a total of 38 novel genomic simple sequence repeats could be identified. The g-SSRs had the motif groups ranging from monomer to pentamer out of which dimer group occurred the most (89.47%). Out of 38 g-SSRs markers developed, 26 were found polymorphic, which showed substantial genetic diversity among the guava genotypes including wild species. The average number of alleles per locus, major allele frequency, gene diversity, expected heterozygosity and polymorphic information content of 26 SSRs were 3.46, 0.56, 0.53, 0.29 and 0.46, respectively. The rate of cross-species transferability of the developed g-SSR loci varied from 38.46 to 80.77% among the studied wild Psidium species. Generation of N-J tree based on 26 SSRs grouped the 40 guava genotypes into six clades with two out-groups, the wild guava species showed genetic distinctness from cultivated genotypes. Furthermore, population structure analysis grouped the guava genotypes into three genetic groups, which were partly supported by PCoA and N-J tree. Further, AMOVA and PCoA deciphered high genetic diversity among the present set of guava genotypes including wild species. Thus, the developed novel g-SSRs were found efficient and informative for diversity and population structure analyses of the guava genotypes. These developed novel g-SSR loci would add to the new genomic resource in guava, which may be utilized in genomic-assisted guava breeding.

Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees].

Kalmegh (Andrographis paniculata (Burm. F.) Nees) is one of the most important medicinal plants and has been widely explored as traditional medicine. To exploit its natural genetic diversity and initiations of molecular breeding to develop novel cultivars or varieties, developments of genomic resources are essential. Four microsatellite-enriched genomic libraries-(CT)14, (GT)12, (AG)15 and (AAC)8-were constructed using the genomic DNA of A. paniculata. Initially, 183 recombinant colonies were screened for the presence of CT, GT, AG, and AAC microsatellite repeats, out of which 47 clones found positive for the desired simple sequence repeats (SSRs). It was found that few colonies had more than one desirable SSR. Thus, a sum of 67 SSRs were designed and synthesized for their validation among 42 A. paniculata accessions. Out of the 67 SSRs used for genotyping, only 41 were found to be polymorphic. The developed set of g-SSR markers showed substantial genetic variability among the selected A. paniculata accessions, with an average polymorphic information content (PIC) value of 0.32. Neighbor-joining tree analysis, population structure analysis, analysis of molecular variance (AMOVA), and principal coordinate analysis (PCoA) illustrated the considerable genetic diversity among them. The novel g-SSR markers developed in the present study could be important genomic resources for future applications in A. paniculata.

Chemopreventive efficacy of Moringa oleifera pods against 7, 12-dimethylbenz[a]anthracene induced hepatic carcinogenesis in mice.

Oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage in a variety of liver disorders. Hence there is a great demand for the development of agents with potent antioxidant effect. The aim of the present investigation is to evaluate the efficacy of Moringa oleifera as a hepatoprotective and an antioxidant against 7, 12-dimethylbenz[a]anthracene induced hepatocellular damage. Single oral administration of DMBA (15 mg/kg) to mice resulted in significantly (p<0.001) depleted levels of xenobiotic enzymes like, cytochrome P450 and b5. DMBA induced oxidative stress was confirmed by decreased levels of reduced glutathione (GSH) and glutathione-S-transferase (GST) in the liver tissue. The status of hepatic aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) which is indicative of hepatocellular damage were also found to be decreased in DMBA administered mice. Pretreatment with the Moringa oleifera (200 and 400 mg/kg) orally for 14 days significantly reversed the DMBA induced alterations in the liver tissue and offered almost complete protection. The results from the present study indicate that Moringa oleifera exhibits good hepatoprotective and antioxidant potential against DMBA induced hepatocellular damage in mice that might be due to decreased free radical generation.

Chemopreventive effect of hydroethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice.

The present study was conducted to investigate the chemopreventive effects of hydro-ethanolic extract of Euphorbia neriifolia (EN) on N-nitrosodiethylamine (DENA) induced renal cancer in male Swiss albino mice. Animals were pretreated with EN extract (150 and 400 mg/kg body weight; p.o) and butylated hydroxyanisole (BHA) as a standard (0.5% and 1% BHA p.o) both for two week prior to the administration of single dose of DENA (50 mg/kg body weight; p.o). Various in vivo antioxidant biochemical parameters like lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT) were evaluated to determine the reno-protective and antioxidant activity of EN. DENA increased oxidative stress through increase in LPO and decrease in antioxidant enzymes (SOD, and CAT). The EN extract significantly restored the antioxidant enzyme level in the kidney and exhibited significant dose dependant protective effect against DENA induced nephrotoxicity, which can be mainly attributed to the antioxidant property of the extract. This study rationalized the ethno-medicinal use of EN for protection against renal cancer.

Water quality modelling of the river Yamuna (India) using QUAL2E-UNCAS.

This paper describes the utility of QUAL2E as a modelling package in the evaluation of a water quality improvement programme. In this study, QUAL2E was applied to determine the pollution loads in the river Yamuna during its course through the national capital territory of Delhi, India. The study aimed at examining the influence of different scenarios on river water quality. Four different pollution scenarios were analysed besides the 'business as usual' situation. The study revealed that it was necessary to treat the discharge from drains to the river Yamuna and diversion of a substantial load to the Agra canal for further treatment was also essential. It was also established through this study that maintaining a flow rate of more than 10 m(3)/s in the river could also help preserve the river's water quality. To clearly display the model outcomes and demarcate polluted zones in the river stretch, model results were interfaced with a Geographical Information System (GIS) to produce cartographic outputs. In addition, uncertainty analysis in the form of first-order error analysis and Monte Carlo analysis was performed, to realise the effect of each model parameter on DO and BOD predictions. The uncertainty analysis gave satisfactory results on simulated data.