Augmenter of liver regeneration enhances the success rate of fetal pancreas transplantation in rodents

Background. Treatment of fetal pancreas (FP) isografts with insulin- like growth factor-I greatly improves the rate of conversion to euglycemia in diabetic rats. Complete knowledge of other factors that may facilitate the engraftment and function of FP in vivo is still embryonic. Augmenter of liver regeneration (ALR) is a newly described polypeptide growth factor found in weanling rat livers. ALR has trophic effects on regenerating liver. We studied the effects of in situ administration of this agent on FP isografts in rats. Methods. Streptozotocin-diabetic Lewis rats (blood glucose >300 mg/dl) received 16 FP isografts transplanted intramuscularly. ALR was delivered from day 1 through day 14, in doses of 40 or 400 ng/kg/d. Animals were followed for 3 months with serial weights and blood glucose monitoring. These animals were compared with those treated with vehicle alone. Results. Of the group treated with ALR at 40 ng/kg/day for 14 days, 89% (eight of nine) were euglycemic (P=0.0003). Of the group treated with ALR at 400 ng/kg/day for 14 days, 88% (seven of eight) were euglycemic (P=0.0007). Of the group treated with vehicle alone, none of the six were euglycemic. Euglycemia is defined here as glucose<200 mg/dl for 3 days. Pathology of the intramuscular transplant site showed patches of islet tissue embedded in fat. These patches demonstrated insulin immunoreactivity. Conclusions. Diabetes was reversed in a significantly greater proportion of FP + ALR-treated recipients than those animals treated with vehicle alone. Local delivery of growth factors my be used as an adjunct to FP transplantation to improve the rate of success. This in situ model may be useful to further evaluate other soluble factors

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system

Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen

Whole Genome Sequencing and Complete Genetic Analysis Reveals Novel Pathways to Glycopeptide Resistance in Staphylococcus aureus

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge

The Bioinformatics Virtual Coordination Network: An open-source and interactive learning environment

Lockdowns and “stay-at-home” orders, starting in March 2020, shuttered bench and field dependent research across the world as a consequence of the global COVID-19 pandemic. The pandemic continues to have an impact on research progress and career development, especially for graduate students and early career researchers, as strict social distance limitations stifle ongoing research and impede in-person educational programs. The goal of the Bioinformatics Virtual Coordination Network (BVCN) was to reduce some of these impacts by helping research biologists learn new skills and initiate computational projects as alternative ways to carry out their research. The BVCN was founded in April 2020, at the peak of initial shutdowns, by an international group of early-career microbiology researchers with expertise in bioinformatics and computational biology. The BVCN instructors identified several foundational bioinformatic topics and organized hands-on tutorials through cloud-based platforms that had minimal hardware requirements (in order to maximize accessibility) such as RStudio Cloud and MyBinder. The major topics included the Unix terminal interface, R and Python programming languages, amplicon analysis, metagenomics, functional protein annotation, transcriptome analysis, network science, and population genetics and comparative genomics. The BVCN was structured as an open-access resource with a central hub providing access to all lesson content and hands-on tutorials (https://biovcnet.github.io/). As laboratories reopened and participants returned to previous commitments, the BVCN evolved: while the platform continues to enable “a la carte” lessons for learning computational skills, new and ongoing collaborative projects were initiated among instructors and participants, including a virtual, open-access bioinformatics conference in June 2021. In this manuscript we discuss the history, successes, and challenges of the BVCN initiative, highlighting how the lessons learned and strategies implemented may be applicable to the development and planning of future courses, workshops, and training programs

Dialysis-associated peritonitis in children

Peritonitis remains a frequent complication of peritoneal dialysis in children and is the most common reason for technique failure. The microbiology is characterized by a predominance of Gram-positive organisms, with fungi responsible for less than 5% of episodes. Data collected by the International Pediatric Peritonitis Registry have revealed a worldwide variation in the bacterial etiology of peritonitis, as well as in the rate of culture-negative peritonitis. Risk factors for infection include young age, the absence of prophylactic antibiotics at catheter placement, spiking of dialysis bags, and the presence of a catheter exit-site or tunnel infection. Clinical symptoms at presentation are somewhat organism specific and can be objectively assessed with a Disease Severity Score. Whereas recommendations for empiric antibiotic therapy in children have been published by the International Society of Peritoneal Dialysis, epidemiologic data and antibiotic susceptibility data suggest that it may be desirable to take the patient- and center-specific history of microorganisms and their sensitivity patterns into account when prescribing initial therapy. The vast majority of patients are treated successfully and continue peritoneal dialysis, with the poorest outcome noted in patients with peritonitis secondary to Gram-negative organisms or fungi and in those with a relapsing infection