Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature

Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics‐based approach to analyse the proteins secreted by B. anthracis Sterne under host‐specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1‐130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature‐regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.     

Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4-14 months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-type > knockout; paw stimulation with Von Frey fibers, knockout < wild-type; balance beam, knockout mobility < WT; thermal sensitivity to heat (tail flick), knockout < wild-type; and cold, knockout < wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.     

A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic syndrome patients from Pakistan

Background

Mutations in the NPHS1 and NPHS2 genes are among the main causes of early-onset and familial steroid resistant nephrotic syndrome respectively. This study was carried out to assess the frequencies of mutations in these two genes in a cohort of Pakistani pediatric NS patients.

Methods

Mutation analysis was carried out by direct sequencing of the NPHS1 and NPHS2 genes in 145 nephrotic syndrome (NS) patients. This cohort included 36 samples of congenital or infantile onset NS cases and 39 samples of familial cases obtained from 30 families.

Results

A total of 7 homozygous (6 novel) mutations were found in the NPHS1 gene and 4 homozygous mutations in the NPHS2 gene. All mutations in the NPHS1 gene were found in the early onset cases. Of these, one patient has a family history of NS. Homozygous p.R229Q mutation in the NPHS2 gene was found in two children with childhood-onset NS.

Conclusions

Our results show a low prevalence of disease causing mutations in the NPHS1 (22% early onset, 5.5% overall) and NPHS2 (3.3% early onset and 3.4% overall) genes in the Pakistani NS children as compared to the European populations. In contrast to the high frequency of the NPHS2 gene mutations reported for familial SRNS in Europe, no mutation was found in the familial Pakistani cases. To our knowledge, this is the first comprehensive screening of the NPHS1 and NPHS2 gene mutations in sporadic and familial NS cases from South Asia.     

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.     

Biological color stripping: A novel technology for removal of dye from cellulose fibers

This research work was carried out to compare the color stripping efficiency of optimized biological method with the chemical stripping, commonly employed in the textile industries. Knitted fabric dyed with Reactive black B dye in 2, 4 and 6% shades strengths was subjected to chemical and biological stripping processes individually. Biological stripping process was found many fold superior to chemical one. It was noted that shade strength does not showed any pronounced effect on the bursting strength of fabric but biological and chemical treatment affect the quality of fabrics in terms of bursting strength/durability of fabric. White rot fungus Ganoderma lucidum IBL-05 showed good potential for decolorization/color stripping of cotton fabric dyed with Reactive black B under optimized set of conditions. The chemical stripping technology is inferior to biological stripping process regarding the quality of fabric and percent color removal from cotton fabric dyed with Reactive black B dye.     

Modification of cellulosic fabric using polyvinyl alcohol—Part-I: Physicochemical properties

A series of poly(vinyl alcohol) of different commercial grades were prepared and applied onto the surfaces of cotton and blends of cotton/polyester fibers. The molecular structure was confirmed using Fourier Transform Infrared spectroscopy. Physicochemical properties such as viscosity and solid contents (%) were determined and discussed. Factors affecting the performance properties of the finished substrate such as post-treatment with poly(vinyl alcohol) of different grades, concentration and dilutions were studied. Fixation of the poly(vinyl alcohol) onto/or within the cellulose structure is accompanied by the formation of semi-inter-penetrated network structure thereby enhancing the association as well as providing very high stiffness. The results revealed that applications of poly(vinyl alcohol) on the textile fabrics in the finishing processes enables to enhance the stiffness as well as helps to improve its pilling resistance.     

Dissipation monitoring for assessing EGF-induced changes of cell adhesion

Epidermal growth factor (EGF)-induced cell de-adhesion has been implicated as a critical step of normal embryonic development, wound repair, inflammatory response, and tumor cell metastasis. Like many other cellular processes, this cell de-adhesion exhibits a complex, time-dependent pattern. A comprehensive understanding of this process requires a quantitative, real-time assessment of cell-substrate interactions at the molecular level. We employed the quartz crystal microbalance with dissipation monitoring (QCM-D) to successfully track the EGF-induced changes in energy dissipation factor, ΔD, of a monolayer of MCF10A cells in real time. This time-dependent ΔD response correlates well both qualitatively and quantitatively with sequential events of a rapid disassembly, transition, and slow reassembly of focal adhesions of the cells in response to EGF exposure. Based on this strong correlation, we utilized the QCM-D to demonstrate that this dynamic focal-adhesion restructuring is regulated temporally by the downstream pathways of EGFR signaling such as the PI3K, MAPK/ERK, and PLC pathways. Because the QCM-D is a noninvasive technique, this novel approach potentially has a broad range of applications in the fundamental study of cellular processes, such as cell signaling and trafficking and mechanotransduction, and holds promise for drug and biomarker screening.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.