Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 μg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca²⁺ ATPase (PMCA) activity was inhibited while intracellular Ca²⁺ level as well as prostaglandin E₂ levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hz × 4 μT × 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca²⁺ levels in blood lymphocytes subsequently inhibiting PGE₂ biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.     

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.     

Preliminary studies on antihyperglycemic effect of aqueous extract of Brassica nigra (L.) Koch in streptozotocin induced diabetic rats

In the streptozotocin induced diabetic rats treated separately with aqueous, ethanol, acetone and chloroform extracts of the seeds of B. nigra, the increase in serum glucose value between 0 and 1 hr of glucose tolerance test (GTT) was the least (29 mg/dl) in aqueous extract treated animals while it was 54, 44 and 44 mg/dl with chloroform, acetone and ethanol extracts respectively. In further studies carried out with aqueous extract, the effective dose was found to be 200 mg/kg body weight in GTT. Administration of 200 mg/kg body weight of aqueous extract to diabetic animals daily once for one month brought down fasting serum glucose (FSG) levels while in the untreated group FSG remained at a higher value. In the treated animals the increase in glycosylated hemoglobin (HbA1c) and serum lipids was much less when compared with the levels in untreated diabetic controls. These findings suggest that further studies with the aqueous extract of B. nigra seeds on its antidiabetic activity would be useful.     

Intestinotrophic effects of exogenous IGF-I are not diminished in IGF binding protein-5 knockout mice

IGF binding protein-5 (IGFBP-5) modulates the availability of IGF-I to its receptor and potentiates the intestinotrophic action of IGF-I. Our aim was to test the hypothesis that stimulation of intestinal growth due to coinfusion of IGF-I with total parenteral nutrition (TPN) solution is dependent on increased expression of IGFBP-5 through conducting our studies in IGFBP-5 knockout (KO) mice. IGFBP-5 KO, heterozygote (HT) and wild type (WT) male and female mice were maintained with TPN or TPN plus coinfusion of IGF-I [recombinant human (rh)IGF-I; 2.5 mg x kg(-1) x day(-1)] for 5 days. The concentration of IGF-I in serum was 73% greater (P < 0.0001) in mice given TPN + IGF-I infusion compared with TPN alone. IGF-I attenuated the 2-3 g loss of body weight associated with TPN in WT mice, whereas KO and HT mice did not show improvement in body weight with IGF-I treatment. KO and HT mice had significantly greater levels of circulating IGF-I binding proteins (IGFBPs) compared with WT mice. Intestinal growth due to IGF-I was observed in all groups treated with IGF-I based on greater concentrations of protein and DNA in small intestine and colon and significantly greater crypt depth and muscularis thickness in jejunum. Jejunal expression of IGFBP-5 mRNA was greater in WT mice, whereas IGFBP-3 mRNA was greater in KO mice treated with IGF-I. In summary, the absence of the IGFBP-5 gene did not block the ability of IGF-I to stimulate intestinal growth, possibly because greater jejunal expression of IGFBP-3 compensates for the absence of IGFBP-5.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

A database for medicinal plants used in treatment of asthma

The knowledge of most plants used in the treatment of asthma, the plant part which is effective in treatment is confined to very few persons who are engaged in folklore medicine. However, this form of medicine is not very popular. Therefore, it is of considerable interest to ethno-botanical community to understand the plants and the parts used for treatment. Here, we describe AsthmaPlantBase, a database containing information of medicinal plants for treatment of asthma.

Availability     

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.     

Semantic integration to identify overlapping functional modules in protein interaction networks

Background  

The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms.