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Remarkable advancement in DNA sequencing (NGS) technology has made personal genome analysis feasible and affordable. Here we present the whole genome sequencing and analysis of three individuals, two males and one female, from different parts of India. Comparison with the Reference Human Genome and the variant database showed a total of 4.0-4.85 million variants, primarily single nucleotide variants (SNVs), 350-600 K small insertions and deletions (INDELs), and previously unreported novel variants. The analysis of Y-chromosome and mitochondrial haplogroups revealed that the ancestors of the individual arrived on the subcontinent at very different times using distinctly different migration routes. Approximately, 500,000 novel SNPs and about 89,000 novel INDELs have been submitted to the NCBI as novel variants. PCA and Admix analysis revealed that the IHGP03, a Mizoram male from the Northeast region, is strikingly different from the other two Indian genomes. Collectively, the data suggest the complexity of the Indian population admix developed from several distinct waves of human migration over tens of thousands of years.

Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent the Shigella infection and reduce the mortality. Conventional approaches of vaccine development against shigellosis have not resulted in desirable vaccine. As shigellosis may be caused by multiple strains and serotypes, there is a need to develop a multivalent vaccine, capable of providing protection against multiple Shigella strains. To develop broad spectrum vaccine, we had previously derived a pool of conserved epitopes against Shigella by using multiple immunoinformatic tools. In this study, the identified conserved epitopes derived from the Outer Membrane Proteins A and C of Shigella were chemically synthesized, and the EpiMix made up of 5 epitopes coupled to a carrier protein, ovalbumin was developed and validated for its immunogenicity. The intramuscular immunization with EpiMix in Balb/c mice led to increase in EpiMix specific serum IgG, and significant increase in fecal IgA as well as in IL-4, IL-2and IFN-γ levels. Further, the EpiMix immunized mice showed protection when challenged against S. flexneri ATCC 12022 using the intraperitoneal route. Moreover, the analysis of cytokine profile and IFN-γ/IL4 ratio in post Shigella challenge immunized mice suggested the high levels of IFN-γ levels and possible dominance of Th1 response, playing pivotal role in the elimination of Shigella. Collectively, the results demonstrate the immunogenic potential and protective efficacy of the EpiMix in the murine shigellosis model. However, the detailed study and further optimisation of epitopes would substantiate the prospective use of EpiMix as a prophylactic candidate for vaccination.

Highlights • LacVax® OmpA elicited humoral and cellular immune responses in BALB/c mice. • Oral immunization with LacVax® OmpA elicited sIgA, a hallmark of mucosal immunity. • LacVax® OmpA immunized mice were protected against Shigella induced dysenteriae. • First report outlining protective potential of L. lactis based vaccine for Shigella. • IgG, sIgA, high levels of INF-γ and IL-10 correlated with protection from Shigella., Shigellosis is an acute invasive disease of the lower intestine, which afflicts millions of people worldwide with an estimated one million fatalities per annum. Despite of extensive research during the last two decades, a vaccine against multi-drug resistant Shigella is not yet available in the market. To provide a safe, effective and broad-spectrum vaccine against Shigella, we explored food grade bacteria Lactococcus lactis (L. lactis) for the delivery of conserved antigenic protein; Outer membrane protein A (OmpA) to the mucosal sites for effective elicitation of systemic and mucosal immunity. We have previously confirmed the immunogenic potential of recombinant L. lactis expressing OmpA (LacVax® OmpA) in BALB/c mice. In the present study, we have characterized the humoral and cellular immune profile of LacVax® OmpA and assessed its protective efficacy using a newly developed human like murine shigellosis model. The significant increase in OmpA specific serum IgG, fecal sIgA and a Th1 dominant immune response (indicated by high INF-γ/IL-4 ratio) in LacVax® OmpA immunized mice revealed successful activation of humoral and cellular immunity. The LacVax® OmpA immunized animals were also protected from human-like shigellosis when challenged with S. flexneri 2a ATCC 12022. The antigen specific serum IgG, fecal sIgA, INF-γ and IL-10 levels were found to be the significant correlates of protection. Collectively these results suggest that the LacVax® OmpA is a promising prophylactic candidate against shigellosis. However, the protective efficacy of LacVax® OmpA in the higher animals would further strengthen its future application in humans.

The article presents the analysis of whole genome sequence of a Gujarati Indian individual (IHGP01) that was sequenced at 23.05× coverage with a total of 74.93 Gb of sequence data generated using Illumina HiSeq 2000 platform. Variant analysis revealed over 3.9 million single nucleotide variants (SNVs) and about 393,000 small insertions and deletions (InDels) including novel variants. The known variants were analyzed for their health and disease relevance and pharmacogenomic profile. Mitochondrial and Y-chromosome haplogroup analysis clearly indicated arrival on the continent not more than 20,000–25,000 years ago, following the route out of Africa to central Europe, then into Asian continent and subsequent migration to West part of the Indian subcontinent. The current research has added 141,000 novel genetic variations to the human DNA database. Functional analysis and validation of these novel variations and revelation of their role in health and disease will add a newer dimension to understand people of this subcontinent.

The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax® DNA-I) in BALB/c mice. Moreover, using EGFP as a model antigen, we also characterized and compared the immune response elicited by LacVax® DNA-I with other conventional vaccination approaches using protein and naked DNA immunization. The presence of antigen-specific serum IgG and fecal secretory IgA (sIgA) antibodies demonstrated in vivo DNA delivery and immune elicitation potential of the developed LacVax® DNA-I. As compared with intramuscular injection, oral delivery of pPERDBY via L. lactis resulted in a significantly rapid increase in IgG and higher sIgA titers, indicating the immunogenic and immunostimulatory properties of the LacVax® DNA-I. The needle-free immunization with LacVax® DNA-I led to increased production of IL-4, an indicator of Th2 screwed response. To the best of our knowledge, this report for the first time outlines comparison of orally administered LacVax® DNA-I with other conventional vaccination approaches.

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.

Shigellosis, a major cause of mortality and morbidity, requires development of effective intervention strategy for which animal model mimicking human pathology is essential. Among various animal models for shigellosis, mice being more convenient have been used wherein intraperitoneal and intranasal routes are preferred. With the aim to comprehend the comparative pathophysiological indicators, we have examined relatively high and low dose of Shigella flexneri administered through intraperitoneal and intranasal routes in mice. Characterization of these two models along with the resulting pathophysiology of shigellosis adds to our understanding and offers suitable models appropriate to the objectives of the study.

Objective The objective of this study was to evaluate the in vitro antiplasmodial properties against malaria parasite in 15 plants mentioned in Indian traditional medicine texts. Methods In vitro antiplasmodial activity of methanolic extracts obtained from Indian traditional medicinal plants was evaluated on Plasmodium falciparum of FCK2 and INDO strains using schizont maturation inhibition assay and parasite lactate dehydrogenase inhibition assay. Results Methanolic extracts of Adhatoda zeylanica, Embelia ribes, Piper nigrum and Plumbago zeylanica exhibited more than 50% inhibition in both the stains in schizont maturation inhibition assay. Methanolic extracts of seven medicinal plants exhibited antiplasmodial activity at half maximal inhibitory concentration (IC50) Conclusion A. zeylanica and E. ribes were the most promising extracts from this study and deserve further investigation of their antiplasmodial properties.

Systemic drug delivery in schizophrenia is a major challenge due to presence of obstacles like, blood-brain barrier and P-glycoprotein, which prohibit entry of drugs into the brain. Quetiapine fumarate (QF), a substrate to P-glycoprotein under goes extensive first pass metabolism leading to limited absorption thus necessitating frequent oral administration. The aim of this study was to develop QF based microemulsion (ME) with and without chitosan (CH) to investigate its potential use in improving the bioavailability and brain targeting efficiency following non-invasive intranasal administration. QF loaded ME and mucoadhesive ME (MME) showed globule size, pH and viscosity in the range of 29-47nm, 5.5-6.5 and 17-40cP respectively. CH-ME with spherical globules having mean size of 35.31±1.71nm, pH value of 5.61±0.16 showed highest ex-vivo nasal diffusion (78.26±3.29%) in 8h with no sign of structural damage upon histopathological examination. Circular plume with an ovality ratio closer to 1.3 for CH-ME depicted ideal spray pattern. Significantly higher brain/blood ratio of CH-ME in comparison to QF-ME and drug solution following intranasal administration revealed prolonged retention of QF at site of action suggesting superiority of CH as permeability enhancer. Following intranasal administration, 2.7 and 3.8 folds higher nasal bioavailability in brain with CH-ME compared to QF-ME and drug solution respectively is indicative of preferential nose to brain transport (80.51±6.46%) bypassing blood-brain barrier. Overall, the above finding shows promising results in the area of developing non-invasive intranasal route as an alternative to oral route for brain delivery.

Picroside I and II, iridoid glycosides, are the major active markers of roots and rhizomes of Picrorhiza kurroa (family: Scrophulariaceae). The rhizomes of P. kurroa have been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and ailments affecting the liver. Various Ayurvedic and herbal preparations are available in the market which contains P. kurroa e.g. Arogyavadhini vati, Tiktadi kwath , Picrolax capsules and suspension. These preparations are used without any significant pharmacokinetics data. Previously, we have reported that oral bioavailability of picroside I and II is low. Most of the iridoid glycosides are primarily metabolized by intestinal microbial flora. So, it is necessary to determine the metabolic profile of picroside I and II and check the correlation with lower bioavailability. Therefore, this study was designed to check metabolic ( in vitro and in vivo ) profile along with pharmacokinetic profile of picroside I and II. For this, a sensitive and selective LC-ESI-MS method was developed and validated for simultaneous determination of picroside I and II in rat plasma. Chromatographic separations were performed on C18 column. The mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer [90:10 v/v], pH 3.5. In-vitro Metabolic study was performed on rat liver microsomes and primary hepatocytes. In-vivo pharmacokinetic and metabolic profile of picroside I and II was generated after oral administration of Kutkin (mixture of picroside I and II) to Sprague-Dawley rats. Various pharmacokinetic parameters viz . C max , T max , AUC (0−t) were determined. In metabolic study, eight metabolites of picroside I and six metabolites of picroside II were identified in vitro , out of which four metabolites for each picroside I and picroside II were identified in vivo .

The objective of the present investigation was to optimize and develop venlafaxine (VLF) loaded nanostructured lipid carrier (NLC) using QbD and risk assessment approach. Full factorial 3 2 design was applied using two independent variables viz ., X1: Drug to total lipid ratio and X2: Surfactant concentration whereby their effect on dependent variables viz ., Y1: Size, Y2: PDI and Y3: %EE was studied. Compritol 888 ATO and Capmul MCM EP showed highest solubility for VLF as solid and liquid lipid respectively which depicted superior miscibility at solid: liquid lipid ratio of 70:30% w/w. VLF NLC were formulated based on optimized formula obtained from design space which gave experimental values for CQA (Size: 77.08 ± 3.45 nm, PDI: 0.234 ± 0.062 and %EE: 81.33 ± 3.05). FTIR, DSC and XRD studies revealed formation of less ordered crystalline structure of lipid matrix favouring higher encapsulation of VLF. Ex-vivo diffusion study showed higher flux value with VLF NLC (14.2 ± 0.40 μg/cm 2 /h) in comparison to VLF solution (9.62 ± 0.59 μg/cm 2 /h) across goat nasal mucosa. VLF NLC did not show toxicity and nasal mucosa was intact indicating safety for intranasal administration. Overall, QbD approach sounds to be apt for the successful development of VLF NLC.

Diarrhoeal diseases due to Shigellosis account for deaths of ~1.5 million children every year in developing countries. Outer membrane proteins (OMPs) of Gram negative bacteria have been shown to be excellent subunit vaccine candidates against various pathogens. However, effective immune response can be generated using specific immunogenic determinants or peptides instead of whole protein or pathogen. In the present study, we chose six OMPs of Shigella flexneri 2a to predict peptides with good antigenic potential. Various tools were used in a systematic flow to predict B- and T-cell epitopes. Stringent selection criteria were used for epitope screening to ensure generation of both arms of immunity. These epitopes are predicted to be effective against a significantly large population of the diarrhoea afflicted countries in Southeast Asia. Most of the predicted epitopes are located towards the outer exposed region of proteins. The epitopes were docked with respective MHC Class I and II molecules to study peptide–MHC interactions. In conclusion, we have predicted an epitope ensemble against Shigellosis which can be experimentally validated for its immunogenic efficacy. We also propose a systematic workflow for immune-optimization to design effective peptide vaccines.

Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax

N-Acetyltransferase 2 (NAT2) gene is known for its polymorphism. The genetic variations leads to the change in the N-acetylation activity and these differences in the acetylation activity leads to the classification of the population into various groups such as rapid, intermediate and slow acetylators. In the present study, we identified different mutations and alleles by sequencing a stretch of 927 bp which covered exon 2 of NAT2 gene and is reported to have all the allelic variants reported till date. Previously identified mutations and some new allele sub-type were detected for NAT2 gene in the present study. Overall, we were able to classify 94 individuals into two distinct groups as slow and intermediate acetylators based on their genotype. Out of all the reported alleles, NAT2*4, *5, *6, *7 and *12 alleles were found in the studied population (n=94) and two new allele subtype NAT2*5P and NAT2*7C were detected in the studied population which have not been reported earlier. We did not observed any gender differences in the present study based on NAT2 acetylation activity. Keywords: N-Acetyltransferase 2 (NAT2), polymorphisms, slow acetylators, rapid acetylators. African Journal of Biotechnology, Vol 13(51) 4639-4646

Presence of tight junctions in blood brain barrier (BBB) pose a major hurdle for delivery of drug and severely affects adequate therapeutic concentration to reach the brain. In present work, we have selected Rivastigmine hydrogen tartrate (RHT), a reversible cholinesterase inhibitor, which exhibits extensive first-pass metabolism, resulting in limited absolute bioavailability (36%). RHT shows extremely low aqueous solubility and poor penetration, resulting in inadequate concentration reaching the brain, thus necessitating frequent oral dosing. To overcome these problems of RHT, microemulsion (ME) and mucoadhesive microemulsion (MME) of RHT were formulated for brain targeting via intranasal delivery route and compared on the basis of in vivo pharmacokinetics. ME and MME formulations containing RHT were developed by water titration method. Characterization of ME and MME was done for various physicochemical parameters, nasal spray pattern, and in vivo pharmacokinetics quantitatively and qualitatively (gamma scintigraphy studies). The developed ME and MME were transparent having globule size approximately in the range of 53–55 nm. Pharmacokinetic studies showed higher values for Cmax and DTP for intranasal RHT: CH-ME over RHT-ME, thus indicating the effect of chitosan in modulating tight junctions, thereby enhanced paracellular transport of RHT. Gamma scintigraphy and in vivo pharmacokinetic study suggested enhanced RHT concentration, upon intranasal administration of RHT:CH-ME, compare with other groups administered formulations intranasally. These findings suggested the potential of non-invasive intranasal route for brain delivery, especially for therapeutics, facing challenges in oral administration.

Genome variations in the form of single nucleotide polymorphism, INDELs (insertions or deletions) and, duplications, inversions and copy number variations are more widely prevalent than initially predicted. Mitochondrial tumor suppressor gene maps on chromosome 8p, in which exon 4-specific deletion is associated with susceptibility towards breast and various other forms of cancer. In this study, 41 head and neck cancer, 15 breast cancer patients and 280 healthy control individuals were analyzed for mitochondrial tumor suppressor gene copy number variation. The frequencies of wt/wt (homozygous wild-type), wt/Del (heterozygous variant) and Del/Del (homozygous deletion variant) mitochondrial tumor suppressor gene genotypes were found to be 73.3%, 26.7%, and 0% in breast cancer patients, 41.5%, 58.5%, and 0% in head and neck cancer patients and 43%, 57%, and 0% in healthy individuals, respectively. A significant association of the deletion variant with breast cancer (odds ratio = 0.27, 95% confidence interval = 0.08–0.87, P = 0.0207) was found in our population. In addition, the allele and genotype frequency varied significantly (P

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system.

Summary Amongst several human genome variations, copy number variations (CNVs) are considered as an important source of variability contributing to susceptibility to wide range of diseases. Although CNV is scattered for genes throughout the human genome, several of autoimmunity related genes have CN variation and therefore play an important role in susceptibility to autoimmune diseases. The association of the Fc gamma receptor 3B (FCGR3B) gene copy number in autoimmunity is well characterized in various populations studied. The Fc gamma receptor is a low affinity, glycosylphosphatidylinositol-linked receptor for IgG molecule predominantly expressed on human neutrophils. The variable gene copy number of FCGR3B is found to be involved in the impaired clearance of immune complexes, which significantly contribute to the pathogenesis of several autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type-1 diabetes and others. The FCGR3B copy number ranged from 0 to ≥2 copies per diploid genome in other populations, but yet not explored in Indian population. Hence, this study aims to evaluate the variation in the frequency distribution of FCGR3B CNV in Indian population. FCGR3B gene copy number varied significantly when compared to other population of the world. This observation will help us in exploring the potential role of CNV in FCGR3B gene and its association to autoimmune disorders in Indian population.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to irreversible loss of neurons, cognition and formation of abnormal protein aggregates. Rivastigmine, a reversible cholinesterase inhibitor used for the treatment of AD, undergoes extensive first-pass metabolism, thus limiting its absolute bioavailability to only 36% after 3-mg dose. Due to extreme aqueous solubility, rivastigmine shows poor penetration and lesser concentration in the brain thus requiring frequent oral dosing. This investigation was aimed to formulate microemulsion (ME) and mucoadhesive microemulsions (MMEs) of rivastigmine for nose to brain delivery and to compare percentage drug diffused for both systems using in-vitro and ex-vivo study. Rivastigmine-loaded ME and MMEs were prepared by titration method and characterized for drug content, globule size distribution, zeta potential, pH, viscosity and nasal ciliotoxicity study. Rivastigmine-loaded ME system containing 8% w/w Capmul MCM EP, 44% w/w Labrasol:Transcutol-P (1:1) and 48% w/w distilled water was formulated, whereas 0.3% w/w chitosan (CH) and cetyl trimethyl ammonium bromide (as mucoadhesive agents) were used to formulate MMEs, respectively. ME and MMEs formulations were transparent with drug content, globule size and zeta potential in the range of 98.59% to 99.43%, 53.8 nm to 55.4 nm and -2.73 mV to 6.52 mV, respectively. MME containing 0.3% w/w CH followed Higuchi model (r(2) = 0.9773) and showed highest diffusion coefficient. It was free from nasal ciliotoxicity and stable for three months. However, the potential of developed CH-based MME for nose to brain delivery of rivastigmine can only be established after in-vivo and biodistribution study.

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.

Background & objectives: The efficacy and toxicity of a given chemotherapy regimen varies widely among patients due to the inherited variability of genes that are involved in drug metabolism. There are several crucial enzymes identified involving metabolism of 5-fluorouracil (5-FU) and cisplatin, which are polymorphic. We studied head and neck cancer patients (n=23) on 5-FU and cisplatin combination therapy attending a tertiary care cancer research institute in Gujarat, India, to understand the effect of a particular genotype on toxicity. Methods: The patients were genotyped for dihydropyrimidine (DPYD) (85T>C, IVS14+1G>A, 2846A>T, 2194G>A), thymidylate synthase (TYMS) [28bp tandem repeat in the promoter enhancer region (TSER)], methylenetetrahydrofolate reductase (MTHFR) (677C>T, 1298A>C), glutathione S-transferase P1(GSTP1) (Ile105Val), glutathione S-transferase T1 (GSTT1) (null allele) and glutathione S-transferase M1 (GSTM1) (null allele) by multiplex allele-specific PCR and long range PCR. Results: Of the 23 (19 males 4 females, age range 18-16 yr) patients, two had grade 3 and 4 toxicity while the remaining 21 had 0 to 2 grade toxicity after treatment with 5-FU and cisplatin combination therapy. An association between the genotype of GSTM1 (+/- and -/-) and the toxicity of cisplatin (P=0.043) was observed. Interpretation & conclusions: The findings of this preliminary study suggested an association between the variants of GSTM1 and toxicity observed due to cisplatin. Well planned studies on a large sample of head and neck cancer patients need to be conducted to understand the effects of these genetic variants on toxicity and efficacy of anticancer drugs.

Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild-type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial-scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.

Clerodendrum inerme L. (Verbenaceae), commonly known as vanajai or garden quinine is a perennial shrub. Leaves and roots of the plant are used in rheumatism and skin diseases. In Indian classical literature the plant is also reported as a substitute of quinine. Since root of the plant is used as drug, whole plant has to be destroyed; this has resulted in the depletion of the plant population. Present investigation was taken up to establish a protocol for mass production of better quality plant material, using axillary bud multiplication. The protocol will help in ex situ conservation of the plant. Maximum number of multiplied axillary bud was observed in 16 M 6-benzyladenine (BA) with 3% sucrose. After elongation, regenerated micro-shoots were rooted in MS medium in absence of plant growth regulators (PGR). The rooted plantlets showed 100% field survival. The regenerated plants showed similar phytochemical profile as mother plant when compared.Keywords: Clerodendrum inerme, finger print profile, micropropogation

Aims To evaluate the comparative immunogenic potential of food grade Lactococcus lactis (L. lactis) expressing Outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. Methods and Results OmpA of SD-1 was cloned and expressed first in Escherichia coli (E. coli) and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and Immunoblot analysis. Using immobilized metal affinity chromatography (IMAC), OmpA was purified from recombinant E. coli BL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA specific IgG antibodies by enzyme linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and fecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Conclusion Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared to the nasal route. Significance and Impact of the Study Using food grade recombinant L. lactis has implications in the development of a prophylactic against multi-drug resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella. This article is protected by copyright. All rights reserved.

Sulfonylureas are widely used to treat type 2 diabetes, with considerable inter-individual variation in the hypoglycaemic response to sulfonylureas. Genetic variants in the gene encoding for transcription factor-7-like 2 ( TCF7L2 ) have been associated with type 2 diabetes. This study aimed to study the effect of variations in TCF7L2 on therapeutic response to sulfonylureas in Type 2 diabetes mellitus patients. The effect of TCF7L2 rs12255372, rs7903146 and rs4506565 genotypes on glycaemic response was observed in 250 diabetic patients treated with sulfonylureas and sulfonylureas along with metformin. The genotyping tests were done by allele-specific multiplex PCR. Glycated haemoglobin (HbA1c) levels were used as phenotypic marker. 60% of sulfonylurea users did not achieve a target HbA1c levels of ⩽6.5% (48 mmol/mol) (which denotes good control in diabetics). Genotype influenced response to sulfonylureas, with more treatment failure in the TT homozygotes in case of rs12255372 and rs4506565. The GG genotype at rs12255372 favourably influences treatment success with sulfonylurea therapy in patients with type 2 diabetes ( p ⩽ 0.05 ). At rs12255372, 70.5% GT or TT genotype failed to achieve therapeutic target, an absolute difference of 19% compared to GG homozygotes. Our preliminary data show that genetic variation at rs12255372 has a direct correlation with therapeutic success with sulfonylureas in type 2 diabetes, hence paving the way for better treatment outcomes in diabetics.

Thymidylate synthase (TS) is the major target for fluoropyrimidine drugs like 5-Fluorouracil (5-FU). There are polymorphic tandem repeats in the TYMS gene enhancer region (TSER). The number of tandem repeats varies in different populations. The aim of this study was to determine the frequencies of the TSER tandem repeats (rs34743033) and compare the observed frequencies with those of other populations.This study genotyped 350 healthy individuals by Polymerase Chain Reaction (PCR).A novel allele *1 (only a single repeat) was observed in four individuals, the individuals were heterozygous (TSER*1/*2) for TYMS. Another variant rs2853542 affecting the expression of Thymidylate synthase was also analysed. The observed genotype frequencies were compared with frequencies observed in other populations for understanding differences between various population groups. There was a statistically significant difference between Indians and Chinese, Kenyans, Ghanians, African-Americans, Americans of European Ancestry, British, Hungarians, Turkish, Australians and Brazilians.This study identified a novel single repeat in the TYMS gene which might have an impact on the expression of this gene, which needs to be confirmed by functional studies.

The objective of the present investigation was to optimize and develop quetiapine fumarate (QF) loaded chitosan nanoparticles (QF-NP) by ionic gelation method using Box-Behnken design. Three independent variables viz., X1-Concentration of chitosan, X2-Concentration of sodium tripolyphosphate and X3-Volume of sodium tripolyphosphate were taken to investigate their effect on dependent variables (Y1-Size, Y2-PDI and Y3-%EE). Optimized formula of QF-NP was selected from the design space which was further evaluated for physicochemical, morphological, solid state characterization, nasal diffusion and in-vivo distribution for brain targeting following non-invasive intranasal administration. The average particle size, PDI, %EE and nasal diffusion were found to be 131.08±7.45nm, 0.252±0.064, 89.93±3.85% and 65.24±5.26% respectively. Neither toxicity nor structural damage on nasal mucosa was observed upon histopathological examination. Significantly higher brain/blood ratio and 2 folds higher nasal bioavailability in brain with QF-NP in comparison to drug solution following intranasal administration revealed preferential nose to brain transport bypassing blood-brain barrier and prolonged retention of QF at site of action suggesting superiority of chitosan as permeability enhancer. Overall, the above finding shows promising results in the area of developing non-invasive intranasal route as an alternative to oral route for brain delivery.

Quantitative evaluation of phytochemical diversity in Adhatoda vasica Nees populations from five different ecogeographical regions was performed using a highly sensitive, robust and economic high performance thin layer chromatography (HPTLC) procedure. The method was validated for specificity, precision and accuracy as per the guidelines affirmed by International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH). The findings support the existence of distinct natural chemotypes within the species. Two out of the five regions had about three times higher mean vasicine content than the remaining three regions. The comparative phytochemical fingerprint profiles were quite similar except for the concentration variation seen for different alkaloids. As the quinazoline alkaloids are the biologically active compounds in A. vasica, the samples that accumulate high levels of alkaloids seem to be promising for further propagation.

Plant-derived natural products occupy an important position in the area of cancer chemotherapy. Molecules such as vincristine, vinblastine, paclitaxel, camptothecin derivatives, epipodophyllotoxin, and so forth, are invaluable contributions of nature to modern medicine. However, the quest to find out novel therapeutic compounds for cancer treatment and management is a never-ending venture; and diverse plant species are persistently being studied for identification of prospective anticancer agents. In this regard,Andrographis paniculataNees, a well-known plant of Indian and Chinese traditional system of medicines, has drawn attention of researchers in recent times. Andrographolide, the principal bioactive chemical constituent of the plant has shown credible anticancer potential in various investigations around the globe.In vitrostudies demonstrate the capability of the compound of inducing cell-cycle arrest and apoptosis in a variety of cancer cells at different concentrations. Andrographolide also shows potent immunomodulatory and anti-angiogenic activities in tumorous tissues. Synthetic analogues of the compound have also been created and analyzed, which have also shown similar activities. Although it is too early to predict its future in cancer chemotherapy, the prologue strongly recommends further research on this molecule to assess its potential as a prospective anticancer agent.

Primary congenital glaucoma (PCG) is a genetically heterogeneous autosomal recessive disorder, which is an important cause of blindness in childhood. The first known gene, CYP1B1, accounts for a variable proportion of cases in most populations. A second gene, LTBP2, was recently reported in association with a syndrome, in which glaucoma is secondary to lens dislocation. We report on the molecular and clinical profile of 34 families diagnosed as PCG, all originating from the Roma/Gypsy founder population. Comprehensive sequencing analysis revealed a level of heterogeneity unusual for this population, with five CYP1B1 and one ancestral LTBP2 mutation accounting for ∼70% of patients (25 out of 37) and the remainder still unexplained. Homozygosity for the founder LTBP2 p.R299X mutation resulted in a more severe clinical phenotype and poorer outcome despite a markedly higher number of surgical interventions. The genetically homogeneous group of p.R299X homozygotes showed variable phenotypes (presumably also underlying pathogenetic mechanisms), wherein PCG proper with primary dysgenesis of the trabecular meshwork, and Marfan syndrome-like zonular disease with ectopia lentis and later onset secondary glaucoma are two extremes. The spectrum manifestations may occur in different combinations and have a different evolution even within the same sibship or a single patient. Preliminary observations on compounds with mutations in both CYP1B1-LTBP2 suggest that the observed combinations are of no clinical significance and digenic inheritance is unlikely. We provide a population genetics perspective to explain the allelic heterogeneity, comparing the history and geographic distribution of the two major founder mutations – p.R299X/LTBP2 and p.E387K/CYP1B1.

Diabetic nephropathy (DN) is one of the foremost causes of renal failure and a primary cause of diabetes mellitus related death. Previously, we have reported that aqueous extract of Enicostemma littorale has potential antidiabetic activity. In the present study, we have investigated the effect of aqueous extract of E. littorale 1 g/kg, p.o. and swertiamarin 50 mg/kg, p.o. daily for 3 weeks in type 1 DN complications in SD rats. DN was assessed by serum urea, creatinine, lipid profile and water intake levels. Treatment with aqueous extract of E. littorale and swertiamarin significantly decreased serum urea and creatinine and other parameters associated with the development of DN in type 1 diabetic rats. We have also found considerable improvement in histology of glomerular function of aqueous extract of E. littorale and swertiamarin-treated animals.

Moringaceae, which belongs to the Moringa oleifera Lam. family, is a well-known herb used in Asian medicine as an antiallergic drug. In the present study, the efficacy of the n-butanol extract of the seeds of the plant (MONB) is examined against ovalbumin-induced airway inflammation in guinea pigs. The test drugs (MONB or dexamethasone) are administered orally prior to challenge with aerosolized 0.5% ovalbumin. During the experimental period, bronchoconstriction tests are performed, and lung function parameters are measured. The blood and bronchoalveolar lavage fluid are collected to assess cellular content, and serum is used for cytokine (tumor necrosis factor-α, interleukin-4, and interleukin-6) assays. Histamine assays of lung tissue are performed using lung tissue homogenate. The results suggest that in ovalbumin-sensitized model control animals, tidal volume is decreased, respiration rate is increased, and both the total and differential cell counts in blood and bronchoalveolar lavage fluid are increased significantly compared with nonsensitized controls. MONB treatment shows improvement in all parameters except bronchoalveolar lavage tumor necrosis factor-α and interleukin-4. Moreover, MONB treatment demonstrates protection against acetylcholine-induced bronchoconstriction and airway inflammation. These results indicate that MONB has an inhibitory effect on airway inflammation. Thus, MONB possesses an antiasthmatic property through modulation of the relationship between Th1/Th2 cytokine imbalances.

We have investigated the hypolipidemic effects of swertiamarin an active lead isolated from a perennial herb Enicostemma littorale Blume. in high cholesterol fed rats. Swertiamarin (50 and 75 mg/kg) and atorvastatin (50 mg/kg) was given orally daily for seven consecutive day to the high cholesterol feed rats. Serum total cholesterol, triglycerides, low density lipoprotein and very low density lipoprotein were found to be markedly elevated in the high cholesterol fed control rats and these changes were significantly prevented in swertiamarin treated animals. However, there was no significant effect on serum high density lipoprotein level. The 3-hydroxy 3-methyl glutaryl Co A (HMG-Co A) reductase activity was significantly inhibited in swertiamarin and atorvastatin treated groups compared to high cholesterol fed control group. Swertiamarin was also found to increased excretion of fecal bile acid and total sterols compared to control animals. In conclusion our data suggest that swertiamarin possess high antiatherogenic potential and an effective cholesterol lowering agent and inhibition of HMG-Co A reductase may be one of the main mechanisms of hypolipidemic effect of swertiamarin.

Background: Dipyrone (metamizole) is a nonsteroidal anti-inflammatory drug used as an analgesic and antipyretic in both pediatric and adult patients. Dipyrone hydrolyses to 4-methylaminoantipyrine (MAA) in the stomach before absorption. There are several HPLC methods available for analysis of MAA from human plasma but no method has yet been developed on liquid chromatography–mass spectrometry (LC–MS) or LC tandem MS (LC–MS/MS), which are much more specific and sensitive techniques. Methodology: A high-performance LC–MS method for the quantification of 4-methylaminoantipyrine from human plasma has been developed, validated and applied to a pharmacokinetic study of 500 mg oral dose dipyrone. Following liquid–liquid extraction, the analyte was first separated on a reverse phase column using isocratic mobile phase and then analyzed by MS in selected ion monitoring mode using [M+H]+ ions, m/z 218.2 for 4-methylaminoantipyrine and 231.3 for 4-isopropylantipyrine (internal standard). Results: The method exhibited a linear range from 0.2 to10.0 µg/ml when only 100 µl human plasma sample was used. The lower limit of detection was 0.04 µg/ml (160 pg on column). The recovery was 80%. The accuracy and precision were obtained over the calibration curve range and were well within the limits specified under guidelines for bioanalytical method validation. The compound was stable under the experimental conditions. Conclusion: The method was demonstrated to be, simple, sensitive and rapid. It can be easily adopted in laboratories with access to LC–MS or MS/MS and applied to sample analysis in clinical settings where a large number of samples are generated.

Injection diclofenac sodium available at present as a 3-ml formulation is administered intragluteally. Standard needles may not reach the gluteus maximus muscle in many cases, especially in obese patients. Alternative sites like the deltoid have been suggested to be more suitable for these patients. Diclofenac sodium 75 mg/ml was prepared to facilitate intradeltoid administration. The objective of this study was to determine pharmacokinetic parameters and to compare the bioavailability of the new formulation of injection diclofenac sodium (75 mg/ml), given as an intradeltoid injection, with the reference formulation (75 mg/3 ml) given intragluteally.A single-dose, two-way bioequivalence study was carried out in 18 healthy, Indian, male volunteers with a washout period of 5 days. Blood samples were collected up to 6 h after drug administration and analyzed using a prevalidated HPLC method.The mean C(max) and T(max), for the test and reference formulations were 1.95 microg/ml, 1.89 microg/ml and 0.39 h, 0.43 h respectively. AUC values from 0 to last measurable time point 't' observed for test and reference formulations were 3.37 microg.h/ml and 3.28 microg.h/ml respectively. The mean AUC 0 - infinity for test and reference formulations was 3.57 microg.h/ml and 3.52 microg.h/ml.Results suggest that the test and reference formulations of injection diclofenac sodium 75 mg given intradeltoid and intragluteally are bioequivalent. The test formulation would be especially helpful in the management of obese/overweight patients and patients with thick subcutaneous fat in the gluteal region. It would increase the success rate of intramuscular deposition of the medication.

Background: Asthma and allergic rhinitis are symptomatically as well as pathophysiologically overlapping conditions and their prevalence is increasing at an alarming rate. Despite significant progress in pathophysiology of asthma and allergic rhinitis and availability of several therapies in recent years, a true and complete cure for asthma and allergic rhinitis so far seems out of reach. Objective: To review clinical efficacy & tolerability, pharmacoeconomics, quality of life, outcomes of several clinical trails based on combination therapy for asthma and allergic rhinitis. Methods: DATA SOURCES: Computer-assisted MEDLINE searches for articles on phathophysiology, clinical trials on combination therapy for asthma and allergic rhinitis, treatment guidelines. Selection: Published articles and pertinent abstracts (1981-2006) on the topics identified above were selected. Results: The pathophysiology of asthma and allergic rhinitis is a complex cascade of events and mono therapy with drugs, which inhibits synthesis and release of a mediator or blocks the receptors of respective mediators, does not result in complete relief in the symptoms. From several clinical trials it can be concluded that combination therapy with two or more drugs may provide additive or synergistic benefits in blocking pathophysiological endpoints and also complement each other by blocking a broad spectrum of inflammatory processes than that provided by either drug alone. Conclusion: From the available scientific data from clinical trials it can be concluded that combination therapeutic approach may be a viable option for the treatment of asthma and allergic rhinitis.

Substituted thiazoles with different structural features were synthesized and screened for their anti-inflammatory activity in acute carrageenin induced rat paw edema model and chronic formalin induced rat paw edema model. The compounds 1-5 showed 83, 30, 63, 69 and 73% protection, respectively, in acute carrageenin induced rat paw edema model. In 5-day chronic formalin induced rat paw edema model on the fifth day 1 and 5 gave 66 and 41% protection. Both studies were carried out at a dose of 100mg/kg body weight. The activity was compared with that of Ibuprofen, Rofecoxib, and Dexamethasone both in acute and chronic anti-inflammatory models. Compound 1 without COX-1 and COX-2 inhibitory activity showed good activity profile almost mimicking the gold standard Dexamethasone in terms of efficacy. A 7-day study in rats at dose of 100mg/kg showed that this compound does not have any ulcerogenic activity and toxicity. The activity of 5 shows that incorporating two pharmacophoric features in one molecule can be a good drug designing strategy. 2,4-Diaminothiazoles with an aliphatic oxime esters attached via a ketone bridge to the 5th position of thiazole was identified as a novel scaffold for designing anti-inflammatory agents.

Gabapentin is an anticonvulsant drug used for the treatment of epilepsy. It is not bound to plasma protein and is not metabolized. A high performance liquid chromatography–mass spectrometric micro method is described in this report for its determination from human plasma. Chromatography was performed on a 50 × 4.6 mm, 4 μm nitrile column and the parent ion detected in the positive ionization mode on single quadrupole analyzer (Q1MI) with atmospheric pressure ionization source. Extraction was carried out on C18, 100 mg/3cc cartridge using 10 μL sample volume. The mean extraction recovery was 97% and within batch and between batch coefficients of variation were

A simple, precise and sensitive high performance liquid chromatography procedure has been developed for determination of carvedilol in human plasma. The method was developed on Lichrosphere R CN column using a mobile phase of acetonitrile/20 mM ammonium acetate buffer with 0.1% triethylamine (pH adjusted to 4.5) (40/60, v/v). The peaks were detected by using fluorescence detector (excitation wavelength 282 nm and emission wavelength 340 nm). Carvedilol and domperidone (internal standard) were extracted by liquid-liquid extraction procedure using dichloromethane. This method was specific and had a linearity range of 1-128 ng/ml with intra- and inter-day precision (%C.V.) less than 15%. The accuracy ranges from 87.3 to 100.88% and the recovery of carvedilol was 69.90%. The stability studies showed that carvedilol in human plasma was stable during short-term period for sample preparation and analysis. This method was used to assay the carvedilol in human plasma samples obtained from subjects who had been given an oral tablet of 12.5 mg carvedilol.

Pitavastatin belongs to the class of coenzyme A reductase inhibitors. Very few methods of assaying pitavastatin from human plasma are available in literature. An analytical method is presented for the determination of the drug from human plasma making use of the fluorescent property of the drug. The drug is extracted from plasma using ethyl acetate under neutral condition and then analyzed by reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection (lambda(Ex) 245 nm; lambda(Em) 420 nm). Analysis of pitavastatin was carried out on a C18 HPLC column using a gradient flow of mobile phase (0.01 mol/L monobasic potassium phosphate (pH 3.20) -acetonitrile, 63:37, v/v). Fluorescein isothiocyanate was used as internal standard. The dynamic range of assay was 3 to 50 ng/mL. The intraday precision was less than 10% and accuracy ranged from 95.2% to 112.6%. The same for interday check was less than 12% and 92.8% to 105.1%, respectively. The drug was found to be stable under the assay conditions. The developed method is simple, precise, accurate, and stable. This indicates that it can be applied to routine analysis of this drug in human subjects where there are large numbers of samples without the need of specialized instruments like column switching.