Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.     

Reaction of α, ω-dicarboxy polyamides in the melt: synthesis of a model

Summary The synthesis of N-dodecanoyl-11-aminoundecanoic acid as a model for carboxylic polyamides 11 is described. The solubilization reaction of these polyamides with trifluoroacetic anhydride is studied by ¹H and ¹³C NMR spectroscopy. The resulting soluble trifluoroacetylated compounds are stable enough to allow an easy GPC study in CH₂Cl₂ on routine instument and columns. The participation of the residual dodecanoic acid in the polycondensation equilibrium of carboxylic polyamides has been established.     

Three-Dimensional Numerical Modeling of Mixing at River Confluences

Lateral mixing of a pollutant is considered as a slow process that is usually complete within 100–300 river widths. Recent studies on flow dynamics at river confluences revealed that lateral mixing can be markedly enhanced when the tributary channel is shallower than the main channel. This study uses a three-dimensional model to examine mixing processes immediately downstream of confluences as well as further downstream in the mainstream. Simulations are presented for a concordant and discordant laboratory junction and a field confluence for a low and a high flow condition. The decrease in standard deviation at a cross section of a tracer over a distance of 5 channel widths is 30% for discordant beds but only 10% for concordant beds in the laboratory simulation. At the natural site, bed discordance is more important at the low flow than at the high flow with corresponding decreases in the standard deviation of 31 and 18% over 3.5 channel widths. Mixing is completed after a distance of 25 and 37 channel widths for the low and high flow conditions, respectively. Further downstream, mixing is mainly affected by planform curvature of the channel.     

Influence of Genetic Variations on Levels of Inflammatory Markers of Healthy Subjects at Baseline and One Week after Clopidogrel Therapy; Results of a Preliminary Study

We aimed to assess the association between the most common polymorphisms of cytochrome P450 (CYP) epoxygenases on the plasma levels of inflammatory markers in a population of healthy subjects. We also sought to determine whether CYP2C19*2 polymorphism is associated with the anti-inflammatory response to clopidogrel. In a population of 49 healthy young males, the baseline plasma levels of inflammatory markers including C-reactive protein, haptoglobin, orosomucoid acid, CD-40 were compared in carriers vs. non-carriers of the most frequent CYP epoxygenase polymorphisms: CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C8*2 and CYP2J2*7. Also, the variation of inflammatory markers from baseline to 7 days after administration of 75 mg per day of clopidogrel were compared in carriers vs. non-carriers of CYP2C19* allele and also in responders vs. hypo-responders to clopidogrel, determined by platelet reactivity tests. There was no significant association between epoxygenase polymorphisms and the baseline levels of inflammatory markers. Likewise, CYP2C19* allele was not associated with anti-inflammatory response to clopidogrel. Our findings did not support the notion that the genetic variations of CYP epoxygenases are associated with the level of inflammatory markers. Moreover, our results did not support the hypothesis that CYP2C19*2 polymorphism is associated with the variability in response to the anti-inflammatory properties of clopidogrel.     

The relationship between fatty acid peroxidation and α-tocopherol consumption in isolated normal and transformed hepatocytes

The response of normal and transformed rat hepatocytes to oxidative stress was investigated. Isolated normal rat hepatocytes and differentiated hepatoma cells (the Fao cell line was derived from the Reuber H 35 rat hepatoma) in suspension were incubated with the ADP/Fe³⁺ chelate for 30 min at 37°C. Membrane lipid oxidation was assessed by measuring (i) free malondialdehyde (MDA) production by a high-performance liquid chromatography (HPLC) procedure, (ii) membrane fatty acid disappearance as judged by capillary gas chromatography, and (iii) α-tocopherol oxidation as determined by HPLC and electrochemical detection. The addition of iron led to increased MDA production in normal as well as in transformed cells, and to simultaneous consumption of polyunsaturated fatty acids (PUFA) and α-tocopherol. In addition, in Fao cells more α-tocopherol was consumed during lipid peroxidation while less PUFA was oxidized. Lipid peroxidation was lower in tumoral hepatocytes than in normal cells. This could be due to a difference in membrane lipid composition because of a lower PUFA content and a higher α-tocopherol level in Fao cells. During oxidation, Fao cells produced 1.5 to 2 times less MDA than normal cells, while in the tumoral cells the amount of oxidized PUFA having 3 or more double bonds was 7 to 8 times lower. Therefore, measuring MDA alone as an index of lipid peroxidation did not allow for proper comparison of the membrane lipid oxidizability of transformed cellsvs. the membrane lipid oxidizability of normal cells.     

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G > C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca²⁺-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 µs molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca²⁺ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.     

Autosomal Dominant Gyrate Atrophy-Like Choroidal Dystrophy Revisited: 45 Years Follow-Up and Association with a Novel C1QTNF5 Missense Variant

We present a long-term follow-up in autosomal dominant gyrate atrophy-like choroidal dystrophy (adGALCD) and propose a possible genotype/phenotype correlation. Ophthalmic examination of six patients from two families revealed confluent areas of choroidal atrophy resembling gyrate atrophy, starting in the second decade of life. Progression continued centrally, reaching the fovea at about 60 years of age. Subretinal deposits, retinal pigmentation or choroidal neovascularization as seen in late-onset retinal degeneration (LORD) were not observed. Whole genome sequencing revealed a novel missense variant in the C1QTNF5 gene (p.(Q180E)) which was found in heterozygous state in all affected subjects. Haplotype analysis showed that this variant found in both families is identical by descent. Three-dimensional modeling of the possible supramolecular assemblies of C1QTNF5 revealed that the p.(Q180E) variant led to the destabilization of protein tertiary and quaternary structures, affecting both the stability of the single protomer and the entire globular head, thus exerting detrimental effects on the formation of C1QTNF5 trimeric globular domains and their interaction. In conclusion, we propose that the p.(Q180E) variant causes a specific phenotype, adGALCD, that differs in multiple clinical aspects from LORD. Disruption of optimal cell-adhesion mechanisms is expected when analyzing the effects of the point mutation at the protein level.     

Evidence of the CH···O HydrogenBonding in Imidazolium-Based Ionic Liquids from Far-Infrared Spectroscopy Measurements and DFT Calculations

Knowledge of all the intermolecular forces occurring in ionic liquids (ILs) is essential to master their properties. Aiming at investigating the weaker hydrogen bonding in aprotic liquids, the present work combined computational study and far-infrared spectroscopy on four imidazolium-based ILs with different anions. The DFT calculations of the ionic couples, using the ωB97X-D functional and considering both the empirical dispersion corrections and the presence of a polar solvent, show that, for all samples, the lowest energy configurations of the ion pair present H atoms, directly bound to C atoms of the cation and close to O atoms of the anion, capable of creating moderate to weak hydrogen bonding with anions. For the liquids containing anions of higher bonding ability, the absorption curves generated from the calculated vibrational frequencies and intensities show absorption bands between 100 and 125 cm⁻¹ corresponding to the stretching of the hydrogen bond. These indications are in complete agreement with the presently reported temperature dependence of the far-infrared spectrum, where the stretching modes of the hydrogen bonding are detected only for samples presenting a moderate interaction and become particularly prominent at low temperatures. Moreover, from the analysis of the infrared spectra, the occurrence of various phase transitions as a function of temperature was detected, and the difference in the average energy between the H-bonded and the dispersion-governed molecular configurations was evaluated.