Immunoinformatic Approach to Contrive a Next Generation Multi-Epitope Vaccine Against Achromobacter xylosoxidans Infections.

Achromobacter xylosoxidans, previously identified as Alcaligenes xylosoxidans, is a rod-shaped, flagellated, non-fermenting Gram-negative bacterium that has the ability to cause diverse infections in humans. As a part of its intrinsic resistance to different antibiotics, Achromobacter spp. is also increasingly becoming resistant to Carbapenems. Lack of knowledge regarding the pathogen's clinical features has led to limited efforts to develop countermeasures against infection. The current study utilized an immunoinformatic method to map antigenic epitopes (Helper T cells, B-cell and Cytotoxic-T cells) to design a vaccine construct. We found that 20 different epitopes contribute significantly to immune response instigation that was further supported by physicochemical analysis and experimental viability. The safety profile of our vaccine was tested for antigenicity, allergenicity, and toxicity against all the identified epitopes before they were used as vaccine candidates. The disulfide engineering was carried out in an area of high mobility to increase the stability of vaccine proteins. In order to determine if the constructed vaccine is compatible with toll-like receptor, the binding affinity of vaccine was investigated via molecular docking approach. With the in silico expression in host cells and subsequent immune simulations, we were able to detect the induction of both arms of the immune response, i.e., humoral response and cytokine induced response. To demonstrate its safety and efficacy, further experimental research is necessary.

Risk of Malignancy in Breast FNAB Categories, Classified According to the Newly Proposed International Academy of Cytology (IAC) Yokohama System.

Background: A new category system comprising five classes (C1-insufficient material, C2-benign, C3-atypical, C4-suspicious, and C5-malignant) has been proposed by the International Academy of Cytology (IAC) for fine needle aspiration biopsy cytology (FNAB) for proper diagnosis of breast cancer. Aims and Objectives: This study is designed to categorize institutional FNAB data according to the new system and calculation of the absolute risk of malignancy (ROM), sensitivity, specificity, positive predictive values, false negative and false-positive rate. Study Design: We conducted a retrospective cross-sectional study involving 2133 cases collected between June, 2008 and August, 2019, at Foundation University Medical College's Department of Histopathology and the Surgery and Oncology Department at the Fauji Foundation Hospital. All cases fulfilling the inclusion and exclusion criteria were retrieved from the archives and reviewed by two expert pathologists. Matching histopathology was compared with the cytology reports for concordance or discordance of results. Findings: We found 6.9% (n = 147) insufficient, 65.8% (n = 1403) benign, 7.2% (n = 153) atypical, 7.5% (n = 160) suspicious and 12.6% (n = 270) malignant cases. Cyto-histological correlation was found in 421 cases from the year 2014 to 2019 with 370 concordant and 51 discordant cases. The maximum number of concordant cases was 151 in the C5 category and discordant cases had a diagnosis of C3 and C4 on cytology with 16 cases in each category. The calculated values of ROM were 45.45%, 10.3%, 30.6%, 82.79% and 99.34% from C1 to C5, respectively. We calculated 83.42% absolute sensitivity and 85.24% specificity. The positive predictive value for category 3, 4 and 5 was 67.34%, 82.7% and 99.34%, respectively, while false-negative rate was 7.9% and false-positive rate was 0.66%. Conclusion: The ROM for C1 category calculated from this study is quite high (45.45%) compared to previous studies; therefore, it is recommended to perform core needle biopsy in all these cases. The higher sensitivity and specificity of this method of diagnosing malignant lesions supports its use.

Pds5A and Pds5B Display Non-redundant Functions in Mitosis and Their Loss Triggers Chk1 Activation.

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-Chk1S317, 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 Chk1S317 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 Chk1S317.

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

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.

Isolation and Antibiogram of Clostridium tetani from Clinically Diagnosed Tetanus Patients.

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.

Phase variable genes of Campylobacter jejuni exhibit high mutation rates and specific mutational patterns but mutability is not the major determinant of population structure during host colonization.

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.