Increased metacyclogenesis of antimony-resistant Leishmania donovani clinical lines

Mathematical models predict that the future of epidemics of drug-resistant pathogens depends in part on the competitive fitness of drug-resistant strains. Considering metacyclogenesis (differentiation process essential for infectivity) as a major contributor to the fitness of Leishmania donovani, we tested its relationship with pentavalent antimony (SbV) resistance in clinical lines. Different methods for the assessment of metacyclogenesis were cross-validated: gene expression profiling (META1 and SHERP), morphometry (microscopy and FACS), in vitro infectivity to macrophages and resistance to complement lysis. This was done on a model constituted by 2 pairs of reference strains cloned from a SbV-resistant and -sensitive isolate. We selected the most adequate parameter and extended the analysis of metacyclogenesis diversity to a sample of 20 clinical lines with different in vitro susceptibility to the drug. The capacity of metacyclogenesis, as measured by the complement lysis test, was shown to be significantly higher in SbV-resistant clinical lines of L. donovani than in SbV-sensitive lines. Together with other lines of evidence, it is concluded that L. donovani constitutes a unique example and model of drug-resistant pathogens with traits of increased fitness. These findings raise a fundamental question about the potential risks of selecting more virulent pathogens through massive chemotherapeutic interventions.     

Binding of N-trifluoroacetyl-derivatized natural glycosphingolipids by uropathogenic Escherichia coli and Neisseria subflava

Several neutral glycosphingolipids were hydrogenated and subjected to trifluoroacetylation in trifluoroacetic acid/trifluoroacetic acid anhydride under conditions leading to complete exchange of the N-acetyl groups of GalNAc for N-trifluoroacetyl. The derivatized glycosphingolipids were analyzed for binding by P-fimbriated uropathogenic Escherichia coli, recognizing the globo series of glycolipids (carrying Gal1-4Gal). Using E. coli it was shown that a GalNCO-CF3 next to the minimum binding epitope Gal1-4Gal did not substantially influence the binding, as did not a trifluoro acetyl group on the ceramide. Exchange of N-acetyl of GalNAc in the receptor active gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer, for N-trifluoroacetyl, did not change the binding of two out of the three strains tested of the bacterium Neisseria subflava. Discussion concerning the binding epitopes of the bacterial adhesins to carbohydrates is based on these results.     

An interlaboratory comparison of ITS2-PCR for the identification of yeasts,using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems

Background  

Currently, most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity. This approach has quite often limited discriminatory power and may require long incubation periods. Due to the increase of fungal infections and due to specific antifungal resistence patterns for different species, accurate and rapid identification has become more important. Several molecular techniques have been described for fast and reliable identification of yeast isolates, but interlaboratory exchangeability of identification schemes of molecular techniques has hardly been studied. Here, we compared amplified ITS2 fragment length determination by an ABI Prism 310 (Applied Biosystems, Foster City, Ca.) capillary electrophoresis system with that obtained by a CEQ8000 (Beckman Coulter, Fullerton, Ca.) capillary electrophoresis system.     

Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains: structure and cross-binding properties

Helicobacter pylori is a bacterium that colonizes the stomach of a majority of the global human population causing common gastric diseases like ulcers and cancer. It has an unusually complex pattern of binding to various host glycoconjugates including interaction with sialylated, sulfated, and fucosylated sequences. The present study describes an additional binding epitope comprising the neolacto internal sequence of GlcNAcbeta3-Galbeta4GlcNAcbeta. The binding was detected on TLC plates as an interaction with a seven-sugar ganglioside of rabbit thymus. The glycolipid was purified and characterized as Neu5Gcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3-Galbeta4Glcbeta1Cer with less than 10% of the fraction carrying a repeated lacto (type-1) core chain, Galbeta3Glc-NAcbeta3Galbeta3GlcNAcbeta. After stepwise chemical and enzymatic degradation and structural analysis of products the strongest binder was found to be the pentaglycosylceramide GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer, whereas the hexa- and tetraglycosylceramides were less active, and the trihexosylceramide was inactive. Further studies revealed that the terminal GlcNAcbeta of the pentaglycosylceramide may be exchanged for either GalNAcbeta3, GalNAcalpha3, or Galalpha3 without loss of the activity. Calculated minimum energy conformers of these four isoreceptors show a substantial topographical similarity suggesting that this binding is a result of a molecular mimicry. Although the glycoconjugate composition of human gastric epithelial cells is not known in detail it is proposed that repeating N-acetyllactosamine units of glycoconjugates may serve as bacterial attachment sites in the stomach.     

Increased angiotensin II type-2 receptor density in hyperplasia,DCIS and invasive carcinoma of the breast is paralleled with increased iNOS expression

Binding of angiotensin II on the angiotensin II type-1 receptor induces cell growth, while triggering via the angiotensin II type-2 (AT(2)) receptor causes an opposing effect of growth inhibition and apoptosis. AT(2) receptor stimulation has also been shown to enhance inducible nitric oxide synthase (iNOS) expression, an enzyme associated with cancer. To study the involvement of the angiotensin II receptors and iNOS in the carcinogenesis of human breast, we visualised both factors in tissues from patients with hyperplasia, ductal carcinoma in situ (DCIS) and invasive carcinoma using immunocytochemistry and in situ hybridisation. In normal ducts, levels for AT(2) protein and mRNA are low, but these are markedly increased in all pathological tissues. While in normal tissue both negative and positive ducts are found, the staining patterns in hyperplasia, DCIS and invasive carcinoma have a homogeneous positive appearance. Similarly, iNOS enzyme expression was very low in the ductal epithelium of normal tissues, but highly increased in all pathologies, with the highest expression found in hyperplastic ducts. Three human cell lines were assayed for the presence of AT(2) receptor. Normal HMec 1001-3 cells were weakly positive, but only one of the adenocarcinoma cell lines, designated SK-BR-3, was shown to express both AT(2) protein and its mRNA. We show that AT(2) receptor and iNOS overexpression are associated with breast disease, further confirming the involvement of the components of the renin-angiotensin system in the aetiology of breast cancer.     

Analysis of the Preserved Amino Acid Bias in Peptide Profiles of Iron Age Teeth from a Tropical Environment Enable Sexing of Individuals Using Amelogenin MRM

The first dental proteomic profile of Iron Age individuals (ca. 2000–1000 years B.P.), collected from the site of Long Long Rak rock shelter in northwest Thailand is described. A bias toward the preservation of the positively charged aromatic, and polar amino acids is observed. It is evident that the 212 proteins identified (2 peptide, FDR <1%) comprise a palimpsest of alterations that occurred both ante‐mortem and post‐mortem. Conservation of amino acids within the taphonomically resistant crystalline matrix enabled the identification of both X and Y chromosome linked amelogenin peptides. A novel multiple reaction monitoring method using the sex specific amelogenin protein isoforms is described and indicate the teeth are of male origin. Functional analysis shows an enrichment of pathways associated with metabolic disorders and shows a capacity for harboring these conditions prior to death. Stable isotope analysis using carbon isotopes highlights the strongly C₃ based (≈80%) diet of the Long Long Rak cemetery people, which probably comprised rice combined with protein from freshwater fish among other food items. The combination of proteomics and stable isotope analysis provides a complementary strategy for assessing the demography, diet, lifestyle, and possible diseases experienced by ancient populations.     

Retrospective analysis of endocarditis patients to investigate the eligibility for oral antibiotic treatment in routine daily practice

Background

According to the current guidelines of the European Society of Cardiology, patients with left-sided infective endocarditis are treated with intravenous antibiotics for 4–6 weeks, leading to extensive hospital stay and high costs. Recently, the Partial Oral Treatment of Endocarditis (POET) trial suggested that partial oral treatment is effective and safe in selected patients. Here, we investigated if such patients are seen in our daily clinical practice.

Methods

We enrolled 119 adult patients diagnosed with left-sided infective endocarditis in a retrospective, observational study. We identified those that would be eligible for switching to partial oral antibiotic treatment as defined in the POET trial (e.g. stable clinical condition without signs of infection). Secondary objectives were to provide insight into the time until each patient was eligible for partial oral treatment, and to determine parameters of longer hospital stay and/or need for extended intravenous antibiotic treatment.

Results

Applying the POET selection criteria, the condition of 38 patients (32%) was stable enough to switch them to partial oral treatment, of which 18 (47.3%), 8 (21.1%), 9 (23.7%) and 3 patients (7.9%) were eligible for switching after 10, 14, 21 days or 28 days of intravenous treatment, respectively.

Conclusion

One-third of patients who presented with left-sided endocarditis in routine clinical practice were possible candidates for switching to partial oral treatment. This could have major implications for both the patient’s quality of life and healthcare costs. These results offer an interesting perspective for implementation of such a strategy, which should be accompanied by a prospective cost-effectiveness analysis.

     

Absence/presence calling in microarray-based CGH experiments with non-model organisms

Structural variations in genomes are commonly studied by (micro)array-based comparative genomic hybridization. The data analysis methods to infer copy number variation in model organisms (human, mouse) are established. In principle, the procedures are based on signal ratios between test and reference samples and the order of the probe targets in the genome. These procedures are less applicable to experiments with non-model organisms, which frequently comprise non-sequenced genomes with an unknown order of probe targets. We therefore present an additional analysis approach, which does not depend on the structural information of a reference genome, and quantifies the presence or absence of a probe target in an unknown genome. The principle is that intensity values of target probes are compared with the intensities of negative-control probes and positive-control probes from a control hybridization, to determine if a probe target is absent or present. In a test, analyzing the genome content of a known bacterial strain: Staphylococcus aureus MRSA252, this approach proved to be successful, demonstrated by receiver operating characteristic area under the curve values larger than 0.9995. We show its usability in various applications, such as comparing genome content and validating next-generation sequencing reads from eukaryotic non-model organisms.     

PPARalpha and PPARgamma regulation of liver and adipose proteins in obese and dyslipidemic rodents

Zucker fatty rats and ob/ob mice are both frequently used hyperlipidemic and insulin-resistant spontaneous genetic models of obesity. We used them to study the effect of PPAR agonists on the protein-expression level in liver and white adipose tissue. PPARalpha-agonist treatments of the rats resulted in that 27% of the quantified hepatic proteins were altered; implicating pronounced peroxisome proliferation and increase in capacity for beta-oxidation of fatty acids although no correction of plasma triglycerides were obtained. On treatment with PPARgamma agonists, adipose proteins were regulated to a much larger extent in the rats compared to mice, 18% and 2%, respectively.