Phase variation of surface structures occurs in diverse bacterial species due to stochastic, high frequency, reversible mutations. Multiple genes of Campylobacter jejuni are subject to phase variable gene expression due to mutations in polyC/G tracts. A modal length of nine repeats was detected for polyC/G tracts within C. jejuni genomes. Switching rates for these tracts were measured using chromosomally-located reporter constructs and high rates were observed for cj1139 (G8) and cj0031 (G9). Alteration of the cj1139 tract from G8 to G11 increased mutability 10-fold and changed the mutational pattern from predominantly insertions to mainly deletions. Using a multiplex PCR, major changes were detected in ‘on/off’ status for some phase variable genes during passage of C. jejuni in chickens. Utilization of observed switching rates in a stochastic, theoretical model of phase variation demonstrated links between mutability and genetic diversity but could not replicate observed population diversity. We propose that modal repeat numbers have evolved in C. jejuni genomes due to molecular drivers associated with the mutational patterns of these polyC/G repeats, rather than by selection for particular switching rates, and that factors other than mutational drift are responsible for generating genetic diversity during host colonization by this bacterial pathogen.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5΄CCCGA and 5΄CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.

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Abstract. Clostridium tetani, the etiologic agent of tetanus, produces a toxin that causes spastic paralysis in humans and other vertebrates. This study was aimed for isolation, identification, and determination of antimicrobial susceptibility of C. tetani from clinically diagnosed tetanus patients. Isolation was done from deep-punctured tissues of the foot and arm injuries of 80 clinically diagnosed tetanus patients from the Pakistan Institute of Medical Sciences hospital. We successfully screened out five C. tetani isolates out of 80 samples based on the strain-specific characteristics confirmed through biochemical testing and toxin production. A disc diffusion method was used for antimicrobial susceptibilities and C. tetani isolates showed susceptibility to cefoperazone, chloramphenicol, metronidazole, penicillin G, and tetracycline, but were found to be resistant to erythromycin and ofloxacin. During animal testing, all the infected mice developed symptoms of tetanus. The results showed that identification of C. tetani is possible using biochemical and molecular tools and that the strains of C. tetani isolated had not developed resistance against the antibiotics most often used for the treatment of tetanus.

In the present study, a co-culture technique was adopted with an aim to investigate a hyper production of exoglucanase, and β-glucosidase using cheap and easily available agro-industrial residue corn stover as growth supporting substrate. Various physio-chemical and nutritional variables were optimized using classical and completely randomized designs for induced production of exoglucanase, and β-glucosidase from the co-culture of Trichoderma viride and Ganoderma lucidum in solid state fermentation (SSF). Analysis profile showed that when the conditions of the SSF medium containing 15 g corn stover substrate (50% w/w moisture) inoculated with 6 mL of inoculum were optimal, the maximum productions of exoglucanase (485 ± 6.5 U/mL) and β-glucosidase (255 ± 3.3 U/mL) were recorded after 5 days of incubation at pH 6 and 35˚C.

Background: Pds5 is an abundant HEAT-repeat-containing protein that binds to cohesin and mediates sister chromatid cohesion. In vertebrates, Pds5A and Pds5B are known to protect DNA replication fork, as their loss leads to DNA damage. Pds5 interacts directly with Wapl, to remove cohesin during mitosis. Aim: To analyze the effects of the loss of Pds5 proteins-mediated DNA damage on the cell cycle checkpoints and to examine the possibility that Pds5 proteins have an overlapping function. Methods: We first analyzed the cell cycle regulation of Pds5 proteins and defects in S-phase; DNA damage was confirmed after Pds5A/B knockdown. The activation of cell cycle checkpoints and apoptosis were examined by the level of p-Chk1 S317 , MAD2 localization, and the level of pro-apoptotic markers, respectively. Results: Pds5 proteins dissociated from chromatin in a stepwise manner, and their loss led to activation of pro-apoptotic markers associated with the phosphorylation of Chk1 S317 due to DNA damage. Depletion of either Pds5A or Pds5B alone increased Smc3 acetylation in perturbed cell cycle, while depletion of both proteins severely impaired Smc3 acetylation. Moreover, the loss of Pds5A/Pds5B activated the SAC in an ATR-Chk1-dependent manner and stabilized Wapl on chromatin. The depletion of Chk1 rescued the S-phase delay associated with Pds5 depletion and significantly increased mitotic catastrophe. Conclusion: Pds5A and Pds5B display overlapping functions in facilitating Smc3 acetylation. Somewhat paradoxically, they also have non-redundant functions in terms of cohesin removal due to the activated surveillance mechanism that leads to phosphorylation of Chk1 S317 .

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Chronic skin wounds and surgical sutures need critical care and fast recovery, robust connection of blood vessels and effective restoration of circulation is necessary in progressive wound healing. Heparin is well known for its' anticoagulant properties, VEGF activation and antithrombosis action. Chitosan aid in the development of the vascular grafts due to its ECM like properties of blood vessels. For electrospinning of Heparin negatively charged and low molecular weight positively charges chitosan, the homogenous solution is required, they precipitate out during mixing due to opposite charges. In the current study, a an efficient strategy is developed for the electrospinning of chitosan and heparin in the presence of lysozyme. The insolubility/non‐homogenous solution formation for electrospinning from charged chitosan and heparin fast acting was solved by using a small amount of N‐cetyl‐N,N,N‐trimethyl ammonium bromide (CTAB) and the resulting solution produced very smooth nanofibers. Polycaprolactone (PCL) was found to be a suitable polymer for the electrospinning of chitosan and heparin using organic and inorganic solvents. Surface morphology of the synthesized fibers was investigated by scanning electron microscopy (SEM) and the presence of functional groups was investigated by FTIR. Degradation studies were performed which revealed that lysozyme loaded materials were degraded much faster as compared to other materials. The angiogenic and biocompatible potential of heparin with 1 mg/ml and 4 mg/ml lysozyme concentration was demonstrated by chorionic allantoic membrane (CAM) assay and it was estimated that 1 mg/ml (lysozyme) loaded CS/HA material was found to be an efficient biomaterial to stimulate angiogenesis.