Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Adaptation of a stable L-form of Bacillus subtilis to minimal salts medium without osmotic stabilizers.

An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes. This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine. The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl.     

Comparison of Campylobacter jejuni genotypes from dairy cattle and human sources from the Matamata-Piako District of New Zealand

Aims: To identify the prevalence and types of Campylobacter jejuni carried by dairy cattle and the extent of overlap of these types with those causing disease in humans. Methods and Results: Faecal samples from 410 dairy cattle were collected from 36 farms in the Matamata-Piako district in New Zealand. Campylobacter jejuni was isolated on all 36 farms, with a prevalence of 51% (95% CI 45–57) in dairy cattle and 65% (95% CI 58–72) in calves. Eighty-nine of these isolates were typed using Penner serotyping and pulsed field gel electrophoresis and were compared with 58 human C. jejuni isolates from people resident within this study area. Conclusions: Campylobacter jejuni were found in the faeces of over half of the dairy cows and calves examined. Twenty-one per cent of the bovine isolates and 43% of the human isolates formed indistinguishable clusters of at least one bovine and one human isolate. Significance and Impact of the Study: While a direct link between bovine isolates and human cases was not demonstrated, the finding of indistinguishable genotypes among C. jejuni isolates from bovine and human sources confirms that dairy cows and calves are a potential source of human campylobacteriosis. Barriers to separate bovine faecal material from the general public are therefore important public health measures.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Quirks of dye nomenclature. 9. Fluorescein

Adolf Baeyer announced the discovery of fluorescein in 1871 and named it after its most striking property, i.e., fluorescence. I describe here the synthesis of fluorescein. There are seven molecular species in both the solid state or in solution. I also summarize some of the diverse applications of the dye, both medical and nonmedical, which depend mostly on the facile detection of fluorescein at low concentration. Both animal and human toxicity are examined.     

Application of Artificial Intelligence/Machine Vision & Learning for the Development of a Live Single-cell Phenotypic Biomarker Test to Predict Prostate Cancer Tumor Aggressiveness

To assess the usefulness and applications of machine vision (MV) and machine learning (ML) techniques that have been used to develop a single cell-based phenotypic (live and fixed biomarkers) platform that correlates with tumor biological aggressiveness and risk stratification, 100 fresh prostate samples were acquired, and areas of prostate cancer were determined by post-surgery pathology reports logged by an independent pathologist. The prostate samples were dissociated into single-cell suspensions in the presence of an extracellular matrix formulation. These samples were analyzed via live-cell microscopy. Dynamic and fixed phenotypic biomarkers per cell were quantified using objective MV software and ML algorithms. The predictive nature of the ML algorithms was developed in two stages. First, random forest (RF) algorithms were developed using 70% of the samples. The developed algorithms were then tested for their predictive performance using the blinded test dataset that contained 30% of the samples in the second stage. Based on the ROC (receiver operating characteristic) curve analysis, thresholds were set to maximize both sensitivity and specificity. We determined the sensitivity and specificity of the assay by comparing the algorithm-generated predictions with adverse pathologic features in the radical prostatectomy (RP) specimens. Using MV and ML algorithms, the biomarkers predictive of adverse pathology at RP were ranked and a prostate cancer patient risk stratification test was developed that distinguishes patients based on surgical adverse pathology features. The ability to identify and track large numbers of individual cells over the length of the microscopy experimental monitoring cycles, in an automated way, created a large biomarker dataset of primary biomarkers. This biomarker dataset was then interrogated with ML algorithms used to correlate with post-surgical adverse pathology findings. Algorithms were generated that predicted adverse pathology with >0.85 sensitivity and specificity and an AUC (area under the curve) of >0.85. Phenotypic biomarkers provide cellular and molecular details that are informative for predicting post-surgical adverse pathologies when considering tumor biopsy samples. Artificial intelligence ML-based approaches for cancer risk stratification are emerging as important and powerful tools to compliment current measures of risk stratification. These techniques have capabilities to address tumor heterogeneity and the molecular complexity of prostate cancer. Specifically, the phenotypic test is a novel example of leveraging biomarkers and advances in MV and ML for developing a powerful prognostic and risk-stratification tool for prostate cancer patients.     

Screening microorganisms for insulin binding reveals binding by Burkholderia multivorans and Burkholderia cenocepacia and novel attachment of insulin to Aeromonas salmonicida via the A-layer

Exposure to microorganisms is considered an environmental factor that can contribute to Type 1 diabetes. Insulin-binding proteins (IBPs) on microorganisms may induce production of antibodies that can react with the human insulin receptor (HIR) with possible consequences in developing a diabetic autoimmune response against HIR and insulin. The interaction of insulin with microorganisms was studied by screening 45 microbial species for their ability to bind insulin. Binding assays were performed using labelled insulin to identify insulin-binding components on the microorganisms. Burkholderia multivorans and Burkholderia cenocepacia isolated from patients with cystic fibrosis (CF) and the fish pathogen Aeromonas salmonicida were the only strains of those tested, which showed insulin-binding components on their cell surfaces. Further work with A.?salmonicida suggested that the insulin-binding activity of A.?salmonicida is due to the A-layer. A mutant of A.?salmonicida lacking the A-layer showed binding, but at a much reduced rate suggesting another insulin-binding component in addition to the high affinity of the A-protein. Soluble protein lysates were subjected to Western ligand blotting using peroxidase-labelled insulin to detect IBPs. Two positive IBPs were apparent at approximately 30 and 20?kDa in lysates from Burkholderia strains, but no IBP was detected in A.?salmonicida lysates.