Whole exome sequencing of breast cancer (TNBC) cases from India: association of MSH6 and BRIP1 variants with TNBC risk and oxidative DNA damage.

Whole exome sequencing in triple negative breast cancer cases (n = 8) and targeted sequencing in healthy controls (n = 48) revealed BRIP1 rs552752779 (MAF: 75% vs. 6.25%, OR 45.00, 95% CI 9.43-243.32), ERBB2 rs527779103 (MAF: 62.5% vs. 7.29%, OR 21.19, 95% CI 5.11-94.32), ERCC2 rs121913016 (MAF: 56.25% vs. 7.29%, OR 16.34, 95% CI 4.02-70.41), MSH6 rs2020912 (MAF: 56.25% vs. 1.04%, OR 122.13, 95% CI 12.29-2985.48) as risk factors for triple negative breast cancer. Construction of classification and regression tree followed by smart pruning identified MSH6 and BRIP1 variants as the major determinants of TNBC (Triple Negative Breast Cancer) risk. Except for ERBB2, all other genes regulate DNA repair and chromosomal integrity. In TNBC cases, two likely pathogenic variations i.e. NCOR1 rs562300336 and PIM1 rs746748226 were observed at frequencies of 18.75% and 12.5%, respectively. Among the 24 variants of unknown significance, MMP9 rs199676062, SYNE1 rs368709678, AURKA rs373550419, ABCC4 rs11568694 have variant allele frequency ≥ 62.5%. These genes regulate metastasis, nuclear modeling, cell cycle and cellular detoxification, respectively. To conclude, aberrations in DNA mismatch repair, nucleotide excision repair or BRCA1 associated genome surveillance mechanism contribute towards triple negative breast cancer.

Genome-Wide Identification and Analysis of Arabidopsis Sodium Proton Antiporter (NHX) and Human Sodium Proton Exchanger (NHE) Homologs in Sorghum bicolor.

Na⁺ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na⁺/H⁺ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein⁻protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Microarray-based SNP genotyping to identify genetic risk factors of triple-negative breast cancer (TNBC) in South Indian population.

In the view of aggressive nature of Triple-Negative Breast cancer (TNBC) due to the lack of receptors (ER, PR, HER2) and high incidence of drug resistance associated with it, a case-control association study was conducted to identify the contributing genetic risk factors for Triple-negative breast cancer (TNBC). A total of 30 TNBC patients and 50 age and gender-matched controls of Indian origin were screened for 9,00,000 SNP markers using microarray-based SNP genotyping approach. The initial PLINK association analysis (p < 0.01, MAF 0.14-0.44, OR 10-24) identified 28 non-synonymous SNPs and one stop gain mutation in the exonic region as possible determinants of TNBC risk. All the 29 SNPs were annotated using ANNOVAR. The interactions between these markers were evaluated using Multifactor dimensionality reduction (MDR) analysis. The interactions were in the following order: exm408776 > exm1278309 > rs316389 > rs1651654 > rs635538 > exm1292477. Recursive partitioning analysis (RPA) was performed to construct decision tree useful in predicting TNBC risk. As shown in this analysis, rs1651654 and exm585172 SNPs are found to be determinants of TNBC risk. Artificial neural network model was used to generate the Receiver operating characteristic curves (ROC), which showed high sensitivity and specificity (AUC-0.94) of these markers. To conclude, among the 9,00,000 SNPs tested, CCDC42 exm1292477, ANXA3 exm408776, SASH1 exm585172 are found to be the most significant genetic predicting factors for TNBC. The interactions among exm408776, exm1278309, rs316389, rs1651654, rs635538, exm1292477 SNPs inflate the risk for TNBC further. Targeted analysis of these SNPs and genes alone also will have similar clinical utility in predicting TNBC.

Expression of avian influenza virus (H5N1) hemagglutinin and matrix protein 1 in Pichia pastoris and evaluation of their immunogenicity in mice.

The conventional avian influenza vaccines rely on development of neutralizing antibodies against the HA and NA antigens. However, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. The M1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce CD8+ T cells and can contribute to protection against morbidity and mortality from influenza. Hence in an effort to study the immune response of rM1 either alone or in combination with rHA, the hemagglutinin (HA) and matrix protein (M1) of A/Hatay/2004/H5N1 strain of avian influenza were expressed in Pichia pastoris as his-tagged proteins and purified through Ni-NTA chromatography. The His-tag was removed using TEV protease cleavage site and the immunogenicity of purified rHA and rM1 either alone or in combination was determined in mice. One group of mice was immunized with 5 µg of purified rHA, the other group was immunized with rM1, and a third group of mice were immunized with 5 µg of rHA and rM1. All the animals were boosted twice, once on 28 days postimmunization (dpi) and the second on 42 dpi. The immune response was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay. The group of mice immunized with rHA and rM1 together showed significantly higher immune response against rHA and rM1 than mice immunized with either HA or M1 antigens. The addition of rM1 with rHA resulted in increased HI titer in animals immunized with both the antigens. These results suggest that the HA and M1 expressed in P. pastoris can be utilized in combination for the development of faster and cost-effective vaccines for circulating and newer strains of avian influenza and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status.

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10(2.69)-10(4.99) viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 µg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.

Effective strategy for pyramiding three bacterial blight resistance genes into fine grain rice cultivar, Samba Mahsuri, using sequence tagged site markers.

Bacterial leaf blight (BB) of rice is a major disease limiting rice production in several rice growing regions of the world. The pathogen, Xanthomonas oryzae pv oryzae, causing the disease is highly virulent to rice crops and is capable of evolving new races. Breeding efforts to incorporate single BB resistant gene often leads to resistance breakdown within a short period. To overcome such breakdown of resistance and develop germplasm with durable disease resistance, we have introgressed three bacterial blight resistance genes, xa5, xa13, and Xa21 into a fine grain rice variety, Samba Mahsuri, using sequence tagged site (STS) markers linked to these genes. Since the efficiency of the STS markers linked to recessive genes to detect homozygotes is less than 100%, we adopted four different pyramiding schemes to minimize loss of recessive resistance genes in advanced backcross generations. Pyramiding scheme A in which a two-gene Samba Mahsuri pyramid line containing Xa21 and xa5 genes was crossed with the Samba Mahsuri line having xa13 gene alone was found to be most effective in preventing the loss of an important recessive gene xa13. We further demonstrated that there was no yield penalty due to pyramiding of multiple genes into the elite indica rice variety.

Development of pilot scale production process and characterization of a recombinant multiepitope malarial vaccine candidate FALVAC-1A expressed in Escherichia coli.

Among the four human malarial species, Plasmodium falciparum causes most of the mortality associated with malaria. Several approaches are being pursued to develop a suitable malaria vaccine since it may be the most effective weapon to fight against malaria. A highly immunogenic, synthetic protein consisting of 21 epitopes from pre-erythrocytic and blood stages of P. falciparum (FALVAC-1A) was constructed and expressed in Escherichia coli. This vaccine candidate was highly immunogenic and induced protective antibodies in rabbits when produced through lab-scale processes in milligram quantities. In order to take this vaccine candidate for further clinical trial, we optimized the process for industrial scale production and purification. Here we describe various methods used in pilot scale production and characterization of FALVAC-1A. A fed-batch cultivation process in a bioreactor at 10-L scale was optimized to express the protein in high yields as inclusion bodies in E. coli cells with the recombinant plasmids. Methods to solubilize, capture and purify the target protein from the inclusion bodies were optimized and the resultant protein was >95% pure based on SDS-PAGE and RP-HPLC. This protein was then refolded and nativity was confirmed by Far-UV CD spectroscopy. Final purified protein was characterized to estimate yield, purity, mass and confirmed to be free of host cell proteins, nucleic acids and bacterial endotoxins. This study confirms that industrial scale clinical grade FALVAC-1A can be produced in a cost-effective manner for clinical trials.

Validation of betapropiolactone (BPL) as an inactivant for infectious bovine rhinotracheitis (IBR) virus.

Infectious bovine rhinotracheitis (IBR) virus causes vulvovaginitis, abortion and respiratory disease in cows and heifers. Betapropiolactone (BPL) is a disinfectant, effective against bacteria, fungi and viruses. It is also used to prepare inactivated vaccines because it destroys the nucleic acid core of viruses but does not damage the capsid. For the validation of BPL when used as an inactivant, it is more important to assure the quality of inactivating agent and the validity of the inactivation process. In the present study, the inactivation kinetics of IBR virus was determined with different concentration of BPL (1:250, 1:500, 1:1000, 1:1500, 1:2000 and 1:2500) at 4 and 37 degrees C. The result indicated that the BPL at 4 degrees C was able to inactivate the IBR virus within 4, 5 and 12h with the concentration of 1:250, 1:500 and 1:1000, respectively. BPL at 37 degrees C was able to inactivate virus within 30 min with the concentration of 1:250. BPL with the concentration of 1:500 and 1:1000 were able to inactivate the virus within 120 min at 37 degrees C. Based on the kinetic study seven formulations were prepared and a sero conversion study of IBR inactivated vaccine was carried out. Serological response in animals to different formulations did not differ significantly (P>0.05).

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench.

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.

First Report of Tobacco streak virus Infecting Guizotia abyssinica from India.

Guizotia abyssinica (L.f.) Cass. (niger), an important oil seed crop grown in India, is used in foods, paints, soaps, and as an illuminant. During a survey conducted in 2004 to monitor Tobacco streak virus (TSV) in Helianthus annuus L. (sunflower) and Arachis hypogaea L. (groundnut), typical symptoms of leaf and petiole necrosis were observed in niger plants from Karnataka State, India. The field-collected samples reacted with TSV-specific polyclonal antiserum in direct antigen coated (DAC)-ELISA. Indicator host species were mechanically inoculated with extracts from symptomatic leaves and grown under greenhouse conditions. The inoculations resulted in local necrotic lesions on Vigna unguiculata cv. C-152 (cowpea), Gomphrena globosa, and Nicotiana tabacum cv. Xanthi (tobacco) at 3 to 4 days postinoculation (dpi) and systemic mosaic mottling on sunflower and G. globosa at 7 to 9 dpi. To identify the virus at the molecular level, total RNA was isolated (RNeasy kit, Qiagen Inc., Chatsworth, CA) from the virus-inoculated cowpea leaf and used for reverse transcription-PCR using TSV CP (coat protein) specific primers (2). The resulting ~720-bp amplicon corresponding to the CP gene of TSV was cloned into pGem-T vector (Promega, Madison, WI) and sequenced. The resulting sequence of the TSV-niger isolate (TSV-NG) comprised 717 nucleotides encoding 238 amino acid residues of the viral coat protein (GenBank Accession No. DQ864458). Comparison of the sequence with those of other TSV CP gene indicated 98.5 to 99.3% nucleotide and 97.9 to 99.6% amino acid sequence identity with TSV isolates from India (1,2; GenBank Accession Nos. AF505073, AY061930, AY061929, AF515823, AF515824, and AF515825). The sequence of TSV-NG had 89.5 and 80.0% amino acid identity with TSV-WC, type strain from the United States (GenBank Accession No. X00435) and TSV-BR, isolate from Brazil (GenBank Accession No. AY354406), respectively. On the basis of symptoms, transmission, and serological and molecular data, the causal agent of necrosis in niger was identified as a strain of TSV widely prevalent in other oil seed and vegetable crops in India. The new report of Tobacco streak virus infecting niger from India, indicated the expansion of host range among oil seed crops. References: (1) A. I. Bhat et al. Indian J Biotechnol. 1:350, 2002. (2) K. S. Ravi et al. Plant Pathol. 50:800, 2001.

Current status of artemisinin and its derivatives as antimalarial drugs.

Artemisinin is a promising and a potent antimalarial drug, which meets the dual challenge posed by drug-resistant parasites and rapid progression of malarial illness. This review article focuses on the progress achieved during the last years in the production of artemisinin from Artemisia annua. The structure, biosynthesis and analysis of artemisinin and its mode of action are described. The review also focuses on clinical studies, toxicity studies, pharmacokinetics and activity of artemisinin related compounds. The production strategies including organic synthesis, extraction from plants, in vitro cultures and alternative strategies for enhancing the yields are also discussed.

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs.

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.