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We report the 4.79-Mb genome sequence of the "Indian Bison Type" biotype of Mycobacterium avium subsp. paratuberculosis strain S5, isolated from a terminally sick Jamunapari goat at the CIRG (Central Institute for Research on Goats) farm in India. This draft genome will help in studying novelties of this biotype, which is widely distributed in animals and human beings in India.

Background: Over the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP; thus, processing these chemicals using current test batteries could require millions of dollars and decades. A need for increased throughput and efficiency motivated the development of methods using in vitro high throughput screening (HTS) assays to prioritize chemicals for EDSP Tier 1 screening (T1S)., Objective: In this study we used U.S. EPA ToxCast HTS assays for estrogen, androgen, steroidogenic, and thyroid-disrupting mechanisms to classify compounds and compare ToxCast results to in vitro and in vivo data from EDSP T1S assays., Method: We implemented an iterative model that optimized the ability of endocrine-related HTS assays to predict components of EDSP T1S and related results. Balanced accuracy was used as a measure of model performance., Results: ToxCast estrogen receptor and androgen receptor assays predicted the results of relevant EDSP T1S assays with balanced accuracies of 0.91 (p < 0.001) and 0.92 (p < 0.001), respectively. Uterotrophic and Hershberger assay results were predicted with balanced accuracies of 0.89 (p < 0.001) and 1 (p < 0.001), respectively. Models for steroidogenic and thyroid-related effects could not be developed with the currently published ToxCast data., Conclusions: Overall, results suggest that current ToxCast assays can accurately identify chemicals with potential to interact with the estrogenic and androgenic pathways, and could help prioritize chemicals for EDSP T1S assays.

The nanoscale interaction of bacterial cells with solid surfaces is a key issue in biomedicine because it constitutes the first pathogenic event in the complex series of biofilm development on prosthetic devices. We report on an Atomic Force Microscopy study of the interaction of Escherichia coli and Pseudomonas aeruginosa bacterial cells with nanostructured titania thin films with controlled and reproducible nanometer-scale morphology, produced by assembling Ti clusters from the gas phase in a Supersonic Cluster Beam Deposition apparatus. The results demonstrate that bacterial adhesion and biofilm formation are significantly influenced by a pure physical stimulus, that is, the nanoscale variation of surface topography. The increase of nanoscale film roughness promotes bacterial adhesion with respect to flat substrates; remarkably, Pseudomonas aeruginosa cells lose their flagella on nanostructured TiO2 thin films upon adhesion, as opposed to same bacteria onto reference smooth glass substrates. Further, we have observed increased cell biovolume and other biofilm properties on nanostructured substrates in comparison with smooth glasses. These findings suggest that the design of innovative biomaterials with a suitable patterning of biomaterials surfaces can be an effective approach to control the adhesion of microorganisms to in vivo implant surfaces with active biological functionalities.

Paratuberculosis (ParaTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic enteritis of ruminants and may contribute to Crohn's disease in humans. Key features of host immunity to MAP infection include an early pro-inflammatory (Th1-like) response that eventually gives way to a predominant anti-inflammatory (Th2-like) response. Many studies have been conducted to understand the underlying mechanism of misdirected host immune response, however, these studies mainly focused on cattle. The present study is the first attempt to test the hypothesis of shift in Th1 to Th2 like responses during the progression of ParaTB in caprine species (small ruminant). Ten healthy male kids (<6 months old) of the same breed were selected for this study. Of the 10 kids, 6 were experimentally infected with native strain (S5) of MAP ("Indian Bison Type") and the remaining 4 kids were control. Kids were monitored for a period of 12 months post infection (MPI) and were tested for establishment of infection. Expression levels of IFNG, IL2, IL12, IL4, and IL10 genes were estimated before infection and at 4, 8, and 12 MPI in stimulated peripheral blood mononuclear cells (PBMCs) of infected and control kids. The study demonstrated the expression of IFNG and IL2 as classic Th1-like pro-inflammatory signatures; whereas, IL10 exhibited itself as classical Th2-like signature. The study also reports unexpected lowered expression of the IL12 gene simultaneously with increased expression of IFNG, lowered expression of the IL2 gene (compared to IFNG), and suppressed expression of the IL4.

Exposure to an imbalance of nutrients prior to conception and during critical developmental periods can have lasting consequences on physiological processes resulting in chronic diseases later in life. Developmental programming has been shown to involve structural and functional changes in important tissues. The aim of the present study was to investigate whether early life diet has a programming effect on the mammary gland. Wild-type mice were exposed from 2 weeks prior to conception to 6 weeks of age to a regular low-fat diet, or to high-fat diets based on either corn oil or flaxseed oil. At 6 weeks of age, all mice were shifted to the regular low-fat diet until termination at 10 weeks of age. Early life exposure to a high-fat diet, either high in n-6 (corn oil) or in n-3 (flaxseed oil) polyunsaturated fatty acids, did not affect birth weight, but resulted in an increased body weight at 10 weeks of age. Transcriptome analyses of the fourth abdominal mammary gland revealed differentially expressed genes between the different treatment groups. Exposure to high-fat diet based on flaxseed oil, but not on corn oil, resulted in regulation of pathways involved in energy metabolism, immune response and inflammation. Our findings suggest that diet during early life indeed has a lasting effect on the mammary gland and significantly influences postnatal body weight gain, metabolic status, and signaling networks in the mammary gland of female offspring.

Nateglinide has been widely used in the treatment of type-2 diabetics as an insulin secretogoga. A reliable, rapid, simple and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for determination of nateglinide in rabbit plasma. The method was developed on Hypersil BDSC-18 column (250 mm×4.6 mm, 5 mm) using a mobile phase of 10 mM phosphate buffer (pH 2.5) and acetonitrile (35:65, v/v). The elute was monitored with the UV-vis detector at 210 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25-2000 ng/mL. The retention times of nateglinide and internal standard (gliclazide) were 9.608 min and 11.821 min respectively. The developed RP-HPLC method can be successfully applied to the quantitative pharmacokinetic parameters determination of nateglinide in rabbit model.

Nanostructured cubic zirconia is a strategic material for biomedical applications since it combines superior structural and optical properties with a nanoscale morphology able to control cell adhesion and proliferation. We produced nanostructured cubic zirconia thin films at room temperature by supersonic cluster beam deposition of nanoparticles produced in the gas phase. Precise control of film roughness at the nanoscale is obtained by operating in a ballistic deposition regime. This allows one to study the influence of nanoroughness on cell adhesion, while keeping the surface chemistry constant. We evaluated cell adhesion on nanostructured zirconia with an osteoblast-like cell line using confocal laser scanning microscopy for detailed morphological and cytoskeleton studies. We demonstrated that the organization of cytoskeleton and focal adhesion formation can be controlled by varying the evolution of surface nanoroughness.

Background: there is an intimate relation between transition metals and cell homeostasis due to the physiological necessity of metals in vivo. Particularly, iron (ferrous and ferric state) is utilized in many physiological processes of the cell but in excess has been linked with negative role contributing in many neurodegenerative processes., Objective: the aim of this study was to investigate which oxidation state of ionic iron (Ferrous (II) versus Ferric (III)) is more toxic to neuronal cells (SHSY(5)Y)., Materials and Methods: The neuroblastoma (SHSY(5)Y) cells were exposed to varying concentration of ferric and ferrous iron. Morphological studies using immunofluorescence staining and microscopic analysis as confirmed by intracellular glutathione (GSH) test demonstrated oxidative stress to cells in iron microenvironment. In addition, MTT assay was performed to evaluate the viability and metabolic state of the cells., Results: the results showed that ferrous form has significantly higher toxicity compared to the ferric ionic state of higher concentration. In addition, microscopic analysis shows cell fenestration at higher concentrations and swelling at intermediate ferric dosages as demonstrated by atomic force microscopy (AFM). Interestingly, the addition of a differentiation inducing factor, trans-retinoic rcid (RA) retains significant viability and morphological features of the cells irrespective of the ionic state of the iron. AFM images revealed clustered aggregates arising from iron chelation with RA., Conclusions: the results indicate that Fe (II) has more toxic effects on cells. In addition, it could be an interesting finding with respect to the antioxidant properties of RA as a chelating agent for the neurodegenerative therapeutics.

Background: Iron involvement/imbalance is strongly suspected in multiple sclerosis (MS) etiopathogenesis, but its role is quite debated. Iron deposits encircle the veins in brain MS lesions, increasing local metal concentrations in brain parenchyma as documented by magnetic resonance imaging and histochemical studies. Conversely, systemic iron overload is not always observed. We explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes in MS patients., Methods: By the pyrosequencing technique, we investigated 414 MS cases [Relapsing-remitting (RR), n=273; Progressive, n=141, of which: Secondary (SP), n=103 and Primary (PP), n=38], and 414 matched healthy controls. Five SNPs in 4 genes were assessed: hemochromatosis (HFE: C282Y, H63D), ferroportin (FPN1: -8CG), hepcidin (HEPC: -582AG), and transferrin (TF: P570S)., Results: The FPN1-8GG genotype was overrepresented in the whole MS population (OR=4.38; 95%CI, 1.89-10.1; P<0.0001) and a similar risk was found among patients with progressive forms. Conversely, the HEPC -582GG genotype was overrepresented only in progressive forms (OR=2.53; 95%CI, 1.34-4.78; P=0.006) so that SP and PP versus RR yielded significant outputs (P=0.009). For almost all SNPs, MS disability score (EDSS), severity score (MSSS), as well as progression index (PI) showed a significant increase when comparing homozygotes versus individuals carrying other genotypes: HEPC -582GG (EDSS, 4.24±2.87 vs 2.78±2.1; P=0.003; MSSS, 5.6±3.06 vs 3.79±2.6; P=0.001); FPN1-8GG (PI, 1.11±2.01 vs 0.6±1.31; P=0.01; MSSS, 5.08±2.98 vs 3.85±2.8; P=0.01); HFE 63DD (PI, 1.63±2.6 vs 0.6±0.86; P=0.009). Finally, HEPC -582G-carriers had a significantly higher chance to switch into the progressive form (HR=3.55; 1.83-6.84; log-rank P=0.00006)., Conclusions: Polymorphisms in the genes coding for iron binding and transporting proteins, in the presence of local iron overload, might be responsible for suboptimal iron handling. This might account for the significant variability peculiar to MS phenotypes, particularly affecting MS risk and progression paving the way for personalized pharmacogenetic applications in the clinical practice.

The pathogenesis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is complex and has not been completely understood yet. In the present study, we analysed the polymorphism in the exon-2 of the caprine major histocompatibility complex (MHC) Class II DRB region and its association with resistance or susceptibility to JD. A total of 203 Jamunapari goats, which is an Indian endangered breed highly susceptible to JD, kept at a single farm were studied. On the basis of clinical signs, microscopic examination, faecal culture, ELISA and diagnostic PCR, 60 and 143 goats were classified as resistant and susceptible to JD, respectively. PCR-based restriction fragment length polymorphism (PCR-RFLP) with two enzymes, PstI and TaqI, was used to assess variation in the DRB gene(s) in all 203 goats studied. Two di-allelic single nucleotide polymorphisms (SNPs), here referred as 'P' and 'T', were tested. In each of them, three genotypes were found in the group analysed. The minimum allele frequencies (MAFs) were 0.233 and 0.486 for the P and T SNPs, respectively. Statistically significant associations between alleles, individual genotypes and composed genotypes of both SNPs were found. The frequency of p and t alleles, of individual pp and tt and of composed pptt genotypes were significantly higher (P(corr) < 0.001) in the 'resistant' group as compared to the 'susceptible' group, while the P and T alleles were associated with susceptibility (P(corr) < 0.001). In heterozygous genotypes, susceptibility was dominant over resistance. The effects of both SNP on resistance and susceptibility were comparable and composed heterozygous genotypes showed intermediate levels of susceptibility in terms of the odds ratio and P-values calculated., (© 2012 Blackwell Publishing Ltd.)

The contamination of implant devices as a result of biofilm formation through bacterial infection has instigated major research in this area, particularly to understand the mechanism of bacterial cell/implant surface interactions and their preventions. In this paper, we demonstrate a controlled method of nanostructured titanium oxide surface synthesis using supersonic cluster beam depositions. The nanoscale surface characterization using atomic force microscopy and a profilometer display a regulated evolution in nanomorphology and physical properties. X-ray photoelectron spectroscopy analyses display a stoichiometric nanostructured TiO(2) film. Measurement of the water contact angle shows a nominal increase in the hydrophilic nature of ns-TiO(2) films, whereas the surface energy increases with decreasing contact angle. Bacterial species Staphylococcus aureus and Escherichia coli interaction with nanostructured surfaces shows an increase in adhesion and biofilm formation with increasing nanoscale morphological properties. Conversely, limiting ns-TiO(2) film distribution to micro/nanopatterned designed substrates integrated with bovine serum albumin functionalization leads to a reduction in biofilm formations due to a globally decreased bacterial cell-surface interaction area. The results have potential implications in inhibiting bacterial colonization and promoting mammalian cell-implant interactions.

Background: An in vivo comparative evaluation to determine the accuracy of working length between radiographic and electronic apex locators., Aim: The study was aimed at evaluating the accuracy of electronic apex locator, to determine the working length of root canal, and to compare it with the radiographic method of working length determination., Materials and Methods: A total of 20 teeth selected for the study had to go for extraction because of periodontal or orthodontic reasons. Access cavity was prepared and the clinical estimated working length (CEWL) was determined with 10-25 no. K-file. A radiograph was then taken for determining the radiographic estimated working length (REWL). For electronic measurement of root canal, a 10 no. K-file was advanced toward the apex until it reached a 0.5 mm short of apex as shown by the apex locator. After fixing the file with a light cured composite, the tooth was extracted, the tooth surface was then longitudinally grounded using straight fissure diamond bur until the root canal and the tip of the file were visible. The distance of file from the minor constriction was measured with help of stereomicroscope., Statistical Analysis: The chi-square test was used for statistical analysis for this study., Results: The chi-square test where χ² = 21.034 with P = 0.000 indicated that a significant difference exists among the groups. The electronic method showed highest number of cases with the working length at the minor constrictor., Conclusion: The electronic method for determining the working length of root canal was found to be more accurate than the radiographic method.

The present investigation concerns with the development of controlled release tablets of lamivudine using acetylated moth bean starch. The acetylated starch was synthesized with acetic anhydride in pyridine medium. The acetylated moth bean starch was tested for acute toxicity and drug-excipient compatibility study. The formulations were evaluated for physical characteristics like hardness, friability, % drug content and weight variations. The in vitro release study showed that the optimized formulation exhibited highest correlation (R) value in case of Higuchi kinetic model and the release mechanism study proved that the formulation showed a combination of diffusion and erosion process. There was a significant difference in the pharmacokinetic parameters (T(max), C(max), AUC, V(d), T(1/2) and MDT) of the optimized formulation as compared to the marketed conventional tablet Lamivir(®), which proved controlled release potential of acetylated moth bean starch., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new composite cation exchanger, tamarind sulphonic acid (TSA) resin has been synthesized. The chemically modified TSA ion exchange resin has been used for the removal and preconcentration of Zn2+, Cd2+, Fe2+, Co2+ and Cu2+ ions in aqueous solution and effluent from the Laxmi steel plant in Jodhpur, India. This type of composite represents a new class of hybrid ion exchangers with good ion exchange capacity, stability, reproducibility and selectivity for toxic metal ions found in effluent from the steel industry. The characterization of the resin was carried out by determining the ion-exchange capacity, elemental analysis, pH titration, Fourier transform infrared spectra and thermal analysis. The distribution coefficients (K(d)) of toxic metal ions were determined in a reference aqueous solution and the steel plant effluent at different pH values; the absorbency of different metal ions on the TSA resin was studied for up to 10 cycles. The adsorption of different metal ions on TSA resin follows the order: Co2+ > Cu2+ > Zn2+ > Fe2+ > Cd2+. The ion exchange capacity of TSA resin is 2.87%.

Highly substituted sago starch phosphate was synthesized using POCl(3) as cross-linking reagent. Titrimetric and Fourier transform infra red (FT-IR) spectral analysis were used to characterize the substitution. Studying the different factors affecting the reaction parameters showed that the optimal conditions for starch phosphorylation were: 4h reaction time and reagent concentration 1.5% (w/w). The physicochemical properties of cross-linked sago starch (CLSS) were done using Scanning electron micrograph (SEM), X-ray powder diffractometer (XRD and Thermogravimetric analysis (TGA). The results revealed that crystalline nature of native sago starch was transformed after cross-linking. TGA report exhibited higher thermal stability, which makes it suitable for various industrial applications. Swelling behavior showed high swelling at low temperature (30 and 60°C) as compared to high temperature (90°C)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Multiple Sclerosis is a multifactorial disease with several pathogenic mechanisms and pathways. Successful MS management and medical care requires early accurate diagnosis along with specific treatment protocols based upon multifunctional nanotechnology approach. This paper highlights advances in nanotechnology that have enabled the clinician to target the brain and CNS in patient with multiple sclerosis with nanoparticles having therapeutic and imaging components. The multipartite theranostic (thera(py) + (diag)nostics) approach puts forth strong implications for medical care and cure in MS. The current nanotheranostics utilize tamed drug vehicles and contain cargo, targeting ligands, and imaging labels for delivery to specific tissues, cells, or subcellular components. A brief overview of nonsurgical nanorepair advances as future perspective is also described. Considering the potential inflammatory triggers in MS pathogenesis, a multifunctional nanotechnology approach will be needed for the prognosis.

The mechanisms by which environmental toxicants alter developmental processes predisposing individuals to adult onset chronic disease are not well-understood. Transplacental arsenic exposure promotes atherogenesis in apolipoprotein E-knockout (ApoE(-/-)) mice. Because the liver plays a central role in atherosclerosis, diabetes and metabolic syndrome, we hypothesized that accelerated atherosclerosis may be linked to altered hepatic development. This hypothesis was tested in ApoE(-/-) mice exposed to 49 ppm arsenic in utero from gestational day (GD) 8 to term. GD18 hepatic arsenic was 1.2 µg/g in dams and 350 ng/g in fetuses. The hepatic transcriptome was evaluated by microarray analysis to assess mRNA and microRNA abundance in control and exposed pups at postnatal day (PND) 1 and PND70. Arsenic exposure altered postnatal developmental trajectory of mRNA and microRNA profiles. We identified an arsenic exposure related 51-gene signature at PND1 and PND70 with several hubs of interaction (Hspa8, IgM and Hnf4a). Gene ontology (GO) annotation analyses indicated that pathways for gluconeogenesis and glycolysis were suppressed in exposed pups at PND1, and pathways for protein export, ribosome, antigen processing and presentation, and complement and coagulation cascades were induced by PND70. Promoter analysis of differentially-expressed transcripts identified enriched transcription factor binding sites and clustering to common regulatory sites. SREBP1 binding sites were identified in about 16% of PND70 differentially-expressed genes. Western blot analysis confirmed changes in the liver at PND70 that included increases of heat shock protein 70 (Hspa8) and active SREBP1. Plasma AST and ALT levels were increased at PND70. These results suggest that transplacental arsenic exposure alters developmental programming in fetal liver, leading to an enduring stress and proinflammatory response postnatally that may contribute to early onset of atherosclerosis. Genes containing SREBP1 binding sites also suggest pathways for diabetes mellitus and rheumatoid arthritis, both diseases that contribute to increased cardiovascular disease in humans.

SIRT1 belongs to the silent information regulator 2 (Sir2) protein family of enzymes and functions as a NAD(+) -dependent class III histone deacetylase. Here, we examined the anti-multiple myeloma (MM) activity of a novel oral agent, SRT1720, which targets SIRT1. Treatment of MM cells with SRT1720 inhibited growth and induced apoptosis in MM cells resistant to conventional and bortezomib therapies without significantly affecting the viability of normal cells. Mechanistic studies showed that anti-MM activity of SRT1720 is associated with: (i) activation of caspase-8, caspase-9, caspase-3, poly(ADP) ribose polymerase; (ii) increase in reactive oxygen species; (iii) induction of phosphorylated ataxia telangiectasia mutated/checkpoint kinase 2 signalling; (iv) decrease in vascular endothelial growth factor-induced migration of MM cells and associated angiogenesis; and (v) inhibition of nuclear factor-κB. Blockade of ATM attenuated SRT1720-induced MM cell death. In animal tumour model studies, SRT1720 inhibited MM tumour growth. Finally, SRT1720 enhanced the cytotoxic activity of bortezomib or dexamethasone. Our preclinical studies provide the rationale for novel therapeutics targeting SIRT1 in MM., (© 2011 Blackwell Publishing Ltd.)

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53-efficient embryonic fibroblasts compared to p53-deficient cells. These cell lines differed with respect to NADH/NAD(+) balance. This ratio constitutes a driving force for NAD- and NADH-dependent reactions and is inversed upon exposure to Rotenone (complex I inhibitor). Rotenone perturbed the structure of the elongated fibrillar tubulin network and decreased mRNA expression of tubulin genes both suggesting reprogramming and reorganization of the cytoskeleton in both cell lines. These changes were reflected in the abundance of specific mRNA and microRNA (miRNA) species as determined from genome-based analysis. Changes in mRNA and miRNA expression profiles reflected differences in energy utilizing pathways, consistent with the notion that the p53 pathway influences the cellular response to mitochondrial dysfunction and that at least some control may be embedded within specific mRNA/miRNA networks in embryonic cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High-throughput screening (HTS) in the U.S. Environmental Protection Agency (EPA) ToxCast™ project provides vast data on an expanding chemical library currently consisting of > 1,000 unique compounds across > 500 in vitro assays in phase I (complete) and Phase II (under way). This public data set can be used to evaluate concentration-dependent effects on many diverse biological targets and build predictive models of prototypical toxicity pathways that can aid decision making for assessments of human developmental health and disease., Objective: We mined the ToxCast phase I data set to identify signatures for potential chemical disruption of blood vessel formation and remodeling., Methods: ToxCast phase I screened 309 chemicals using 467 HTS assays across nine assay technology platforms. The assays measured direct interactions between chemicals and molecular targets (receptors, enzymes), as well as downstream effects on reporter gene activity or cellular consequences. We ranked the chemicals according to individual vascular bioactivity score and visualized the ranking using ToxPi (Toxicological Priority Index) profiles., Results: Targets in inflammatory chemokine signaling, the vascular endothelial growth factor pathway, and the plasminogen-activating system were strongly perturbed by some chemicals, and we found positive correlations with developmental effects from the U.S. EPA ToxRefDB (Toxicological Reference Database) in vivo database containing prenatal rat and rabbit guideline studies. We observed distinctly different correlative patterns for chemicals with effects in rabbits versus rats, despite derivation of in vitro signatures based on human cells and cell-free biochemical targets, implying conservation but potentially differential contributions of developmental pathways among species. Follow-up analysis with antiangiogenic thalidomide analogs and additional in vitro vascular targets showed in vitro activity consistent with the most active environmental chemicals tested here., Conclusions: We predicted that blood vessel development is a target for environmental chemicals acting as putative vascular disruptor compounds (pVDCs) and identified potential species differences in sensitive vascular developmental pathways.

Environmental Protection Agency's ToxCast project is profiling the in vitro bioactivity of chemicals to assess pathway-level and cell-based signatures that correlate with observed in vivo toxicity. We hypothesized that developmental toxicity in guideline animal studies captured in the ToxRefDB database would correlate with cell-based and cell-free in vitro high-throughput screening (HTS) data to reveal meaningful mechanistic relationships and provide models identifying chemicals with the potential to cause developmental toxicity. To test this hypothesis, we built statistical associations based on HTS and in vivo developmental toxicity data from ToxRefDB. Univariate associations were used to filter HTS assays based on statistical correlation with distinct in vivo endpoint. This revealed 423 total associations with distinctly different patterns for rat (301 associations) and rabbit (122 associations) across multiple HTS assay platforms. From these associations, linear discriminant analysis with cross-validation was used to build the models. Species-specific models of predicted developmental toxicity revealed strong balanced accuracy (> 70%) and unique correlations between assay targets such as transforming growth factor beta, retinoic acid receptor, and G-protein-coupled receptor signaling in the rat and inflammatory signals, such as interleukins (IL) (IL1a and IL8) and chemokines (CCL2), in the rabbit. Species-specific toxicity endpoints were associated with one another through common Gene Ontology biological processes, such as cleft palate to urogenital defects through placenta and embryonic development. This work indicates the utility of HTS assays for developing pathway-level models predictive of developmental toxicity.

Objectives: In this study we aimed to estimate the prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) in animal attendants who were chronic colitis patients or who had inflammatory bowel disease and were suspected for Crohn's disease; these animal attendants worked with goat herds endemic for Johne's disease. Microscopic examination and culture tests were used. For comparison purposes a group of healthy human subjects (not suffering with colitis) was also screened., Methods: Stool samples obtained from 98 human subjects (58 animal attendants suspected for Crohn's disease and 40 healthy humans) were screened for the presence of MAP by microscopic examination and culture. Of the 58 animal attendants screened, 38 had abdominal pain, 29 had suffered episodes of diarrhea, 39 had experienced weight loss, 27 had fever, and 32 had a history of raw milk consumption. Animal attendants had had contact of variable duration with goat herds endemic for Johne's disease (1-5, 6-10, 11-15, and >15 years). Forty stool samples from healthy humans with no symptoms/history of contact with animals were also screened. IS900 PCR and IS1311 PCR restriction endonuclease analysis were used to characterize and genotype the MAP colonies., Results: MAP was recovered from 34 of the 98 human subject stool samples (34.7%). Of the 98 samples, 16.3% (n=16) were acid-fast. None of the 40 healthy human subjects were positive for MAP by microscopy, but five (12.5%) were positive for MAP by culture. Of the 58 animal attendants, 16 (27.6%) were positive by microscopy and 29 (50%) were positive by culture. MAP were recovered from 68.4% of animal attendants with abdominal pain, 72.4% of those with diarrhea, 71.8% of those with weight loss, 44.4% of those with fever, and 46.9% of those who had a history of raw milk consumption. Of the 29 culture-positive animal attendants, 48.3% had worked for >15 years, 27.6% for 11-15 years, 20.7% for 6-10 years, and 3.4% for 1-5 years with goat herds endemic for Johne's disease. Of the 34 culture isolates, 28 (82.4%) showed good quality DNA on agarose gel and were positive by IS900 PCR. Of the 28 IS900-positive DNA samples, 23 (82.1%) were genotyped as 'Indian bison type' and five (17.9%) as 'cattle type'., Conclusions: The prevalence of MAP was higher in attendants suffering from gastrointestinal problems who worked with goat herds endemic for Johne's disease, than in humans with no history of contact with animals. The risk of developing gastrointestinal problems with clinical symptoms indistinguishable from inflammatory bowel disease was higher in humans who were in contact with goat herds endemic for Johne's disease as compared to healthy humans, and the risk was correlated with the duration of association with the endemic goat herds., (Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The relationship of vasculogenic mimicry to pigment in nodular vertical growth phase [VGP] cutaneous melanomas is assessed in this study. 10 nodules each from 27 tumors, 15 pigmented and 12 amelanotic were sampled in proportion to the pigment level. Serial frozen and paraffin sections subjected to HE, Reticulin, PAS to assess the vascular pattern; Dopa Oxidase and Immunopositivity for HMB45, LN5 [laminin 5] & integrin[α(5)β(1)], and EM [electron microscopy] to identify Weibel-Palade bodies within endothelial cells. The vascular pattern, pigment and the immunopositivity was mapped to assess the percentage VM [vasculogenic sinusoids] vs INC [incorporated microvasculature]. In pigmented melanomas, INC from pre-existing stromal vessels is predominant. Amelanotic melanomas show embryonic vasculogenic mimicry, a self-propagating system of spaces within the sheets of tumors cells. Both INC and VM co-exist in tumors with both amelanotic and melanotic nodules. In areas with VM, loci of LN5 and α(5)β(1) integrin positive cells appear within the proliferating columns, positivity in these cells suggesting a switch to a more aggressive form. Irregular spaces appear lined by tumor cells, with initial hemopoeitic activity, coalesce and interlink into tubular networks. Spaces lined by tumor cells extend into an intricate network which then connects with the angiogenetic system. The tumor cells lining the vasculogenic spaces are positive for LN5, α(5)β(1) integrin. Statistically, INC is significantly higher in pigmented melanomas, whereas amelanotic melanomas show significantly higher VM. Pigmentation is correlated positively with INC and negatively with VM. INC and VM are negatively correlated with each other.

Previous studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti-multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.

In present pilot study aimed to estimate, presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in the human serum samples originating from North India using "Indigenous absorbed ELISA kit" (ELISA kit). The phase I, "ELISA kit" was optimized using protoplasmic antigen from native isolate of MAP "Indian Bison type" recovered from the biopsies of Crohn's disease patients. The phase II, sensitivity and specificity of the kit were estimated as 40.0 and 83.3%, respectively, when evaluated in 40 human serum samples (5 Crohn's disease and 22 ulcerative colitis patients and 13 healthy human subjects) with defined MAP status with respect to stool culture. Seroprevalence of MAP antibodies was higher in CD patients (80.0%) as compared to ulcerative colitis patients (4.5%) and normal human subjects (15.3%). The phase III, seroprevalence of MAP antibodies was estimated as 23.4%, on the basis of the screening of 452 human serum samples (without history) from different geographical regions of North India. Region-wise, 34.0, 33.3, 32.8, 25.0, 23.0, 17.7, and 12.5% samples were positive from the states of Punjab, Uttarakhand, New Delhi, Himachal Pradesh, Haryana, Uttar Pradesh and Jammu and Kashmir, respectively. Study reported moderately higher presence of MAP antibodies in human population, which necessitates programs to reduce the bioburden of MAP in the environment and in animal population.

In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as 'Indian Bison type', which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Understanding the potential health risks posed by environmental chemicals is a significant challenge elevated by the large number of diverse chemicals with generally uncharacterized exposures, mechanisms, and toxicities. The present study is a performance evaluation and critical analysis of assay results for an array of 292 high-throughput cell-free assays aimed at preliminary toxicity evaluation of 320 environmental chemicals in EPA's ToxCast™ project (Phase I). The chemicals (309 unique, 11 replicates) were mainly precursors or the active agent of commercial pesticides, for which a wealth of in vivo toxicity data is available. Biochemical HTS (high-throughput screening) profiled cell and tissue extracts using semi-automated biochemical and pharmacological methodologies to evaluate a subset of G-protein coupled receptors (GPCRs), CYP450 enzymes (CYPs), kinases, phosphatases, proteases, HDACs, nuclear receptors, ion channels, and transporters. The primary screen tested all chemicals at a relatively high concentration 25 μM concentration (or 10 μM for CYP assays), and a secondary screen re-tested 9132 chemical-assay pairs in 8-point concentration series from 0.023 to 50 μM (or 0.009-20 μM for CYPs). Mapping relationships across 93,440 chemical-assay pairs based on half-maximal activity concentration (AC50) revealed both known and novel targets in signaling and metabolic pathways. The primary dataset, summary data and details on quality control checks are available for download at http://www.epa.gov/ncct/toxcast/., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In the present study, cross linked sodium carboxymethylated pea starch (SCPS) was synthesized and evaluated as tablet superdisintegrant in diclofenac sodium based tablets. SCPS was synthesized using native pea starch with monochloroacetic acid and NaOH in microwave radiation environment. Finally the dried product was cross-linked with phosphorous oxychloride, which produced granular highly swellable starch. SCPS with degree of substitution of 0.34 was formed and it was further evaluated as superdisintegrant in diclofenac sodium based tablets. Diclofenac sodium tablets were prepared by direct compression method with 2, 4, 6 and 8%w/w of SCPS as superdisintegrant and further comparatively evaluated for in vitro disintegration and dissolution study with Sodium starch glycolate containing tablets as reference. The results revealed that SCPS could be a promising superdisintegrant for immediate release tablets in concentration dependant manner.

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.

PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM., (© 2010 Blackwell Publishing Ltd.)

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.

Fecal samples of 708 domestic and 27 wild ruminants from seven districts of three states submitted to Microbiology Laboratory from 2004 to 2008 were screened for the presence and genotype diversity of Mycobacterium avium subspecies paratuberculosis (MAP) stusing modified microscopic examination and culture tests. MAP colonies were characterized and genotyped by IS900 PCR and IS1311 PCR-REA, respectively. In microscopic examination and culture, 18.7 and 31.2% sampled were positive, respectively. Percent positivity varied in different species and agro-climatic regions. Of the 151 DNA recovered from 230 MAP isolates, 94.7% (143) and 5.2% (8) were genotyped as 'Indian Bison type' and 'Cattle type', respectively. Cattle and buffaloes were infected with both the genotypes, whereas goat and sheep were infected exclusively by 'Indian Bison type'. Study showed low diversity in MAP genotypes and 'Indian Bison type' was the predominant genotype shared by different species, breeds and agro-climatic regions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Surgical trauma, exposure to an external circuit, and reduced organ perfusion contribute to the systemic inflammatory response following cardiopulmonary bypass (CPB). Reduced splanchnic perfusion causes disruption of the gastrointestinal mucosal barrier and the release of endotoxins. Fenoldopam (a new dopamine 1 receptor agonist) has been shown to be a specific renosplanchnic vasodilator in animal and human studies. We studied the effects of fenoldopam on the systemic inflammatory response and the release of endotoxins after CPB and compared the results with those for dopexamine., Methods: Our prospective randomized study included 42 consecutive patients with good to moderate left ventricular function who were to undergo elective or inpatient coronary artery bypass grafting. We used closed envelope method to randomize patients to receive 0.2 μg/kg per minute of fenoldopam (n = 14), 2 μg/kg per minute of dopexamine (n = 14), or normal saline (n = 14). Patients received their respective treatments continuously from anesthesia induction until the end of the first 24 postoperative hours. Interleukin 1β (IL-1β), IL-6, IL-8, IL-10, IL-12, tumor necrosis factor α, complement 3a (C3a), C4a, C5a, and endotoxins were measured during the perioperative period. Repeated-measures analysis of variance was used to evaluate the results for the timed samples., Results: There were no statistical differences between the groups with respect to pre- and intraoperative variables. Release of C3a was attenuated in the fenoldopam group (P = .002), and release of IL-6 and IL-8 was attenuated in the postoperative period in the fenoldopam group (P = .012 and .015, respectively). The other interleukins showed no uniform release in any of the 3 groups. There were no statistically significant differences in serum endotoxin elevation between the 3 groups., Conclusion: A partial attenuation in the inflammatory response is possible with fenoldopam infusion. The elevation in serum endotoxin levels was not affected by dopexamine or fenoldopam infusion.

The iron metallobiology has long been suspected as a causal agent in venous leg ulcer (VLU) pathophysiology. However, it was demonstrated only recently that visible iron deposits cause lesions in only some individuals due to functional iron and related gene variants. In this article, the mechanism by which dysregulated iron cycle leads to local iron overload that could generate free radicals or activate a proteolytic hyperactivity on the part of matrix metalloproteinases (MMPs) or else downregulate tissue inhibitors of MMPs is reviewed. Also reviewed is the interplay of other vital factors such as coagulation factor XIII (FXIII), which influences tissue remodeling and angiogenesis, leading to impaired healing of the lesion, whether there exists altered interaction with MMPs or in presence of particular unfavorable single nucleotide polymorphisms.

Background: Melanomas, highly malignant tumors arise from the melanocytes which originate as multipotent neural crest cells during neural tube genesis. The purpose of this study is to assess the pattern of neural differentiation in relation to angiogenesis in VGP melanomas using the tumor as a three dimensional system., Methods: Tumor-vascular complexes [TVC] are formed at the tumor-stroma interphase, by tumor cells ensheathing angiogenic vessels to proliferate into a mantle of 5 to 6 layers [L1 to L5] forming a perivascular mantle zone [PMZ]. The pattern of neural differentiation is assessed by immunopositivity for HMB45, GFAP, NFP and synaptophysin has been compared in: [a] the general tumor [b] tumor-vascular complexes and [c] perimantle zone [PC] on serial frozen and paraffin sections., Statistical Analysis: ANOVA: Kruskal-Wallis One Way Analysis of Variance; All Pairwise Multiple Comparison Procedures [Tukey Test]., Results: The cells abutting on the basement membrane acquire GFAP positivity and extend processes. New layers of tumor cells show a transition between L2 to L3 followed by NFP and Syn positivity in L4&L5. The level of GFAP+vity in L1&L2 directly proportionate to the percentage of NFP/Syn+vity in L4&L5, on comparing pigmented PMZ with poorly pigmented PMZ. Tumor cells in the perimantle zone show high NFP [65%] and Syn [35.4%] positivity with very low GFAP [6.9%] correlating with the positivity in the outer layers., Discussion: From this study it is seen that melanoma cells revert to the embryonic pattern of differentiation, with radial glial like cells [GFAP+ve] which further differentiate into neuronal positive cells [NFP&Syn+ve] during angiogenic tumor-vascular interaction, as seen during neurogenesis, to populate the tumor substance.

Unlabelled: Reduced organ perfusion during cardiopulmonary bypass (CPB) is responsible for morbidity associated with cardiac surgery. Non-pulsatile flow and hypothermia during CPB have been shown to cause reduced perfusion. During CPB, cardiac output is directly proportional to the pump flow rate. Therefore, we hypothesised that increasing pump flow during hypothermic CPB would improve organ perfusion and reduce the inflammatory response in the post-operative period., Methods: Ethics committee approval was obtained. Twelve consecutive patients with good or moderate left ventricular function undergoing elective or inpatient coronary artery bypass grafting were included in the study after obtaining informed consent. Patients were randomised to receive either normal flow or higher pump flow (20% more than the usual flow during hypothermia). Hepatic blood flow, cytokines such as interleukins 1β, 6, 8, 10 and 12, tumour necrosis factor-α and complements C3a, C4a and C5a were measured during the peri-operative period. Data were analysed using SPSS (ver.15). Categorical data were compared using the chi-square test and trends in cytokines were compared using a repeated measures ANOVA test., Results: Both the groups were similar in pre- and peri-operative variables. Hepatic blood flow almost doubled in the high-pump-flow group following an increase in the flow rate during hypothermia(p=0.026). The release of serum complement IL-6 and 8 appeared to be reduced in the high-flow group; however, the difference did not reach statistical significance., Conclusions: Higher pump flows during hypothermic CPB increase hepatic blood flow. There was a trend towards attenuation of post-operative inflammatory response; however, larger studies will be needed to confirm these findings.

Present study aimed to genotype Mycobacterium avium subspecies paratuberculosis (MAP) recovered from suspected and Crohn' s disease patients. A total of 32 MAP and DNA (directly from clinical samples) recovered from human origin were genotyped using IS 1311 PCR-REA. Isolates were cultured from stool, biopsies and blood clots of Crohn's disease patients, and stool samples of suspected (animal attendants, lab workers etc). Of the 32 MAP isolates belonging to 28 human beings, majority (84.3%) were genotyped as 'Bison type', while 21.7% were of 'cattle' and none was 'sheep' genotype. Study first time reports distribution of 'Cattle' and 'Bison type' 'genotypes in suspected and Crohn's patients on pilot scale in India. 'Bison type' genotype was predominant in the surveyed human population.

Our previous study showed that the novel proteasome inhibitor NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM-xenografts demonstrated that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase-1 clinical trial of NPI-0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI-0052 using human MM xenograft murine model. Our results showed that NPI-0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin-like (CT-L, beta5), trypsin-like (T-L, beta2), and caspase-like (C-L, beta1) activities in extra-vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H-NPI-0052) in mice demonstrated that NPI-0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S-bearing mice with NPI-0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease or paratuberculosis, a gastro intestinal inflammatory condition in ruminants and other animals, which is similar to Crohn's disease (CD) that occurs in man. The role of MAP in the causation of CD has been under intense investigation in the last few decades. This review summarizes the status of MAP in animals and the food chain and its association with CD in man.

Information on Mycobacterium avium subspecies paratuberculosis (MAP) genotypes infecting different animal species in India is limited. Presence of MAP was investigated in free ranging antelopes (locally known as Nilgai/blue bulls/Boselaphus tragocamelus) using direct microscopy, culture, IS900 PCR and IS1311 PCR-REA. IS900 elements of MAP from Nilgai and previously isolated from goats were sequenced and compared to establish inter-species transmission between free ranging Nilgai and closed farm herds and flocks of goats and sheep sharing common grazing and water resources. Fecal samples were collected from two geographical regions (Mathura and Kanpur Dehat districts) separated by 300km, in North India. Of the 42 fecal samples cultured, MAP colonies were recovered from 23.8% samples (Nilgai). Of the 10 positive fecal samples, two were in 'Super shedder' (>1000cfu/g) category and rest were moderate (<10-100cfu/g) shedders. None of the Nilgai from Kanpur Dehat was positive in culture. The 229bp fragment targeting specific IS900 sequence was amplified from template DNA isolated from all the positive MAP cultures of Nilgai. Using IS1311 PCR-REA, MAP colonies were genotyped as 'Bison type'. Goatherds and a sheep flock located at Central Institute for Research on Goats (CIRG), shared 303.52ha of land (Mathura district of Uttar Pradesh) with Nilgai and were endemic for MAP infection. MAP strains isolated from goats and sheep have been genotyped as 'Bison type'. Nucleotide sequence of the insertion elements (900) from MAP 'Bison type' strain (S5) of goat origin and MAP (B42) from Nilgai showed difference of 2 (1%) base pairs at the 11th and 12th position (Genbank accession number EU130943). Study is first report on sharing (inter-species transmission) of a new 'Bison type' genotype of MAP between free ranging wildlife (Nilgai population) and domestic animals (farm goatherds and sheep flocks) in India., (2008 Elsevier Ltd. All rights reserved.)

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in animals, has also been linked with Crohn's disease in human beings. Lack of indigenous diagnostics and vaccine hampered control of JD in India. Designing effective control strategies require thorough understanding of the etiological agent at phenotypic and molecular levels. On the basis of cultural phenotypes and IS1311 PCR-REA typing, MAP strains have been genotyped as 'Cattle type', 'Sheep type' and 'Bison type'. Information exists on genetic differences and comparative evolution of 'Cattle type' and 'Sheep type' strains after divergence from M. avium; however, emphasis has been little on 'Bison type' strains. Recently, a new 'Indian Bison type' genotype has been reported as principal strain infecting different animal species and human beings in India. The study analyzed few genetic markers to have inferences on the molecular evolution of native MAP isolates belonging to 'Bison type' genotype. Results pointed towards recent evolution of 'Bison type' genotype., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Background: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, a chronic enteritis evocative of human inflammatory bowel disease. In industrialized countries MAP has been cultured from pasteurized milk, compounding the increasing concern that MAP may be zoonotic. The purpose of this study was to evaluate commercially available unpasteurized and pasteurized milk and its products for the presence of viable MAP or MAP DNA from an area of northern India with a population of 150 million people., Methods: We studied 43 samples (16 unpasteurized, 27 pasteurized) purchased in Mathura, Agra, or New Delhi, for the presence of MAP by culture or by PCR for IS900 MAP DNA. Positives results were confirmed as MAP by restriction endonuclease analysis and/or DNA sequencing., Results: Colonies appeared in 1.5-20 months post-inoculation. Of the unpasteurized samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were positive for IS900 MAP DNA. Of the pasteurized samples, 67% (18/27) were MAP culture-positive and 33% (9/27) were IS900-positive. Subsequently, 100% (25/25) of the cultured colonies were IS900 and IS1311 MAP DNA-positive., Conclusions: This is the first report from a developing country of MAP cultured from both pasteurized and unpasteurized milk and milk products. Thus we corroborate the presence of viable MAP in the food chain reported from industrialized countries. With the increasing concern that MAP may be zoonotic, these findings have major implications for healthcare in India. The decreased sensitivity in detecting MAP DNA by PCR directly from milk should be ascribed to our employing only one set of PCR primers., (Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

Mycobacterium avium subspecies paratuberculosis (MAP), is the etiological agent of Johne's disease (or paratuberculosis) in animals and has also been linked with Crohn's disease of human beings. Extreme fastidious nature of the organism (MAP) has hampered studies on diversity within the organism. Studies based on phenotypic properties like growth rate, pigmentation, lipid profile etc., are unable to provide complete information on diversity of MAP organism in nature. However, with the advent of molecular assays (IS900 RFLP, PFGE, IS1311 PCR-REA, SSR typing, VNTR typing etc.) in last 2 decades, progress has been made to differentiate MAP strains. MAP isolates have been classified into various types and subtypes using these molecular tools. Optimization of these typing assays has led to generation of new information about MAP strains, subtypes, their comparative genomics, relative evolution, comparative virulence etc. Knowledge of strain diversity is important for better understanding of molecular and sero-epidemiology, infection and patho-biology, vaccine development and planning control strategies. The present review provides available information on MAP strains, ho st adaptations, their virulence,comparative genomics, relative genetic evolution and differentiation.

Efficacy of IS900 blood PCR was evaluated for the presence of MAP infection. Serum, fecal, and blood samples of kids, young, and adult goats from farm and farmer's herds in Mathura district were also screened by ELISA, microscopy and culture. Of 111 goats (kids: 40, young: 14, adults: 57) screened, 77.5% were positive by blood PCR. Of 76 goats, 90.8% (kids: 87.5% and adults: 94.4%) were positive by PCR. From 21 kids and 14 young goats, 42.8 and 57.1% were positive. gDNA from goats was genotyped as MAP "Indian Bison type". Of 21 fecal samples of kids examined by microscopy, 66.7% were positive. In ELISA, 9.5 and 57.1% kids were positives as "type I" and "type II" reactors, respectively. Screening 14 young goats by culture of blood clots, 28.6% were positive. Agreement was substantial between PCR and microscopy. It was fair and moderate when PCR and microscopy were compared with type I and type II reactors, respectively. Presence of MAP in non-clinical kids and young goats indicate early or subclinical infection. Blood PCR was rapid, sensitive, and specific assay for detection of MAP in any stage (early, subclinical, and clinical) and age (kids, young, and adult) of goats.

Therapeutic efficacy of an "Indigenous vaccine" has been evaluated with respect to a commercial vaccine (Gudair, Spain), for the control of clinical Johne's disease (JD) in naturally infected goatherds. Seventy-one goats (JD positive) were randomly divided into 3 groups ("Bison", "Gudair" and "Sham-immunized"). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of M. paratuberculosis (MAP) in feces, internal condition and lesions, as well as humoral and cell-mediated immune responses for 210 days. Study showed marked overall improvement in physical condition of vaccinated goats and average body weight gain was significantly higher (P < .05) in "Bison" group as compared to "Sham-immunized" goats. Mortality due to JD was significantly (P < .05) lower in vaccinated groups than in "sham-immunized". Morbidity rates (due to diarrhea and weakness) were lower in "Bison" group as compared to other groups. Died goats from vaccinated groups showed regression of gross JD lesions and regeneration of fat layer around visceral organs while "Sham-immunized" goats exhibited frank lesions. Vaccinated goats had higher protective CMI response and also higher antibody titer for the trial period as compared to "Sham immunized". Both vaccines also reduced shedding of MAP in feces significantly (P < .05). Though the two vaccines effectively restricted the severity of clinical symptoms of JD, however "Indigenous vaccine" was superior in many respects.

The present study was performed to determine experimental conditions for thalidomide induction of fetal malformations and to understand the molecular mechanisms underlying thalidomide teratogenicity in cynomolgus monkeys. Cynomolgus monkeys were orally administered thalidomide at 15 or 20mg/kg-d on days 26-28 of gestation, and fetuses were examined on day 100-102 of gestation. Limb defects such as micromelia/amelia, paw/foot hyperflexion, polydactyly, syndactyly, and brachydactyly were observed in seven of eight fetuses. Cynomolgus monkeys were orally administered thalidomide at 20mg/kg on day 26 of gestation, and whole embryos were removed from the dams 6h after administration. Three embryos each were obtained from the thalidomide-treated and control groups. Total RNA was isolated from individual embryos, amplified to biotinylated cRNA and hybridized to a custom Non-Human Primate (NHP) GeneChip((R)) Array. Altered genes were clustered into genes that were up-regulated (1281 genes) and down-regulated (1081 genes) in thalidomide-exposed embryos. Functional annotation by Gene Ontology (GO) categories revealed up-regulation of actin cytoskeletal remodeling and insulin signaling, and down-regulation of pathways for vasculature development and the inflammatory response. These findings show that thalidomide exposure perturbs a general program of morphoregulatory processes in the monkey embryo. Bioinformatics analysis of the embryonic transcriptome following maternal thalidomide exposure has now identified many key pathways implicated in thalidomide embryopathy, and has also revealed some novel processes that can help unravel the mechanism of this important developmental phenotype., (Copyright 2009 Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is primarily an autoimmune disorder of unknown origin. This review focuses iron overload and oxidative stress as surrounding cause that leads to immunomodulation in chronic MS. Iron overload has been demonstrated in MS lesions, as a feature common with other neurodegenerative disorders. However, the recent description of chronic cerebrospinal venous insufficiency (CCSVI) associated to MS, with significant anomalies in cerebral venous outflow hemodynamics, permit to propose a parallel with chronic venous disorders (CVDs) in the mechanism of iron deposition. Abnormal cerebral venous reflux is peculiar to MS, and was not found in a miscellaneous of patients affected by other neurodegenerative disorders characterized by iron stores, such as Parkinson's, Alzheimer's, amyotrophic lateral sclerosis. Several recently published studies support the hypothesis that MS progresses along the venous vasculature. The peculiarity of CCSVI-related cerebral venous blood flow disturbances, together with the histology of the perivenous spaces and recent findings from advanced magnetic resonance imaging techniques, support the hypothesis that iron deposits in MS are a consequence of altered cerebral venous return and chronic insufficient venous drainage.