Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles

Little information regarding the composition of the gut microbiota in preterm infants is available. The purpose of this study was to investigate the bacterial diversity in faeces of preterm infants, using analysis of randomly cloned 16S rRNA genes and PCR-TTGE (temporal temperature gradient gel electrophoresis) profiles, to determine whether noncultivated bacteria represented an important part of the community. The 288 clones obtained from faecal samples of 16 preterm infants were classified into 25 molecular species. All but one molecular species had a cultivated representative in public databases: molecular tools did not reveal any unexplored diversity. The mean number of molecular species per infant was 3.25, ranging from one to eight. There was a high interindividual variability. The main groups encountered were the Enterobacteriaceae family and the genera Enterococcus, Streptococcus and Staphylococcus. Seven preterm infants were colonized by anaerobes and only four by bifidobacteria. TTGE profiles were composed of one to nine bands (mean value: 4.3). Furthermore, 51 of 59 clones (86%) comigrated with a band of the corresponding faecal sample. This study will form a comparative framework for other studies, e.g. on the faecal microbiota of preterm infants with different pathologies or the impact of diet on colonization.     

Salicylic acid and its location in response to biotic and abiotic stress

Salicylic acid (SA) is an important signal involved in the activation of plant defence responses against abiotic and biotic stress. SA may derive from the phenylpropanoid pathway or via isochorismate synthase as demonstrated in Nicotiana benthamiana, tomato and Arabidopsis thaliana. The phenylpropanoid pathway as well as isochorismate synthase are localized in the chloroplasts but it remains unknown if the end product SA is in the same organelle. We have studied the localization of SA in A. thaliana using the salicylate hydroxylase (NahG) gene expressed with a chloroplast targeting sequence. Plants expressing NahG in the chloroplasts are unable to accumulate SA induced after pathogen or UV exposure. Our data infer that SA is initially located in the chloroplasts.     

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Summary Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD⁺ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate     

Relationship between Urinary Level of Phytate and Valvular Calcification in an Elderly Population: A Cross-Sectional Study

Pathological calcification generally consists of the formation of solid deposits of hydroxyapatite (calcium phosphate) in soft tissues. Supersaturation is the thermodynamic driving force for crystallization, so it is believed that higher blood levels of calcium and phosphate increase the risk of cardiovascular calcification. However several factors can promote or inhibit the natural process of pathological calcification. This cross-sectional study evaluated the relationship between physiological levels of urinary phytate and heart valve calcification in a population of elderly out subjects. A population of 188 elderly subjects (mean age: 68 years) was studied. Valve calcification was measured by echocardiography. Phytate determination was performed from a urine sample and data on blood chemistry, end-systolic volume, concomitant diseases, cardiovascular risk factors, medication usage and food were obtained. The study population was classified in three tertiles according to level of urinary phytate: low (<0.610 μM), intermediate (0.61–1.21 μM), and high (>1.21 μM). Subjects with higher levels of urinary phytate had less mitral annulus calcification and were less likely to have diabetes and hypercholesterolemia. In the multivariate analysis, age, serum phosphorous, leukocytes total count and urinary phytate excretion appeared as independent factors predictive of presence of mitral annulus calcification. There was an inverse correlation between urinary phytate content and mitral annulus calcification in our population of elderly out subjects. These results suggest that consumption of phytate-rich foods may help to prevent cardiovascular calcification evolution.     

Fine needle aspiration cytology of neurothekeoma. A case report

BACKGROUND: Neurothekeoma (NT) is a rare, benign neoplasm of soft parts with a distinctive histologic appearance. To our knowledge, the cytologic findings have not been described before. We present a case of NT with the cytologic features on fine needle aspiration cytology (FNAC). CASE: A 54-year-old female presented with a circumscribed nodule in the left breast. The lesion was evaluated by FNAC. The smears showed an abundant, metachromatic, myxoid matrix with fusiform and epithelioid cells, some binucleated or multinucleated, loose or in groups and sometimes forming concentric whorls. The lesion was removed, and the diagnosis of NT was made after histopathologic study. CONCLUSION: NT is an extremely rare neoplasm in the mammary region. Fusiform and epithelioid cells arranged in concentric whorls in a myxoid tumor of soft tissue are a distinctive characteristic of this neoplasm and can suggest the diagnosis.     

In silico mutagenesis of human ACE2 with S protein and translational efficiency explain SARS-CoV-2 infectivity in different species

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding.     

Plasma concentrations of pregnancy-associated glycoprotein-1 (PAG-1) in high producing dairy cows suffering early fetal loss during the warm season

The present study was designed to establish whether plasma pregnancy-associated glycoprotein-1 (PAG-1) measurements during the early fetal period can be associated with early fetal loss. Blood samples were obtained and ultrasound controls performed on days 35, 42, 49, 56, and 63 of gestation or until pregnancy loss from 98 lactating dairy cows. Radioimmunoassay systems were used to determine PAG-1 and progesterone concentrations. Of the 98 pregnancies investigated 18 (18.4%) suffered early fetal loss: 15 (18.5%) in cows carrying singletons, and 3 (16.7%) in twin pregnancies. In cows suffering pregnancy loss, all living embryos registered on day 35 seemed normal in size and development in all weekly ultrasound controls before fetal expulsion. Using analysis of variance, plasma PAG-1 and progesterone values were not different between no loss and fetal loss groups for every gestation period. Based on the odds ratio, and considering only PAG-1 values obtained on day 35 of gestation, the risk of fetal loss was 10 and 6.8 times more likely in cows with low (<2.5 ng/ml) and high (>4 ng/ml) PAG-1 values, respectively, than in cows with medium PAG-1 values, used as reference. Of the 10 inseminating bulls included in the study, one was related to increased fetal loss by odds ratio of 21.7, whereas one bull was attributed fetal loss rate reduced by odds ratio of 12.5 (1/0.08) These findings can have a clear clinical application: PAG-1 measurements from one single sample taken on day 35 of gestation provided more useful information than a series of values obtained from day 35 to 63 of gestation, and can be indicators of subsequent fetal loss.     

Model misspecification confounds the estimation of rates and exaggerates their time dependency

While welcoming the comment of Ho et al. ( 2015 ), we find little that undermines the strength of our criticism, and it would appear they have misunderstood our central argument. Here we respond with the purpose of reiterating that we are (i) generally critical of much of the evidence presented in support of the time‐dependent molecular rate (TDMR) hypothesis and (ii) specifically critical of estimates of μ derived from tip‐dated sequences that exaggerate the importance of purifying selection as an explanation for TDMR over extended timescales. In response to assertions put forward by Ho et al. ( 2015 ), we use panmictic coalescent simulations of temporal data to explore a fundamental assumption for tip‐dated tree shape and associated mutation rate estimates, and the appropriateness and utility of the date randomization test. The results reveal problems for the joint estimation of tree topology, effective population size and μ with tip‐dated sequences using beast . Given the simulations, beast consistently obtains incorrect topological tree structures that are consistent with the substantial overestimation of μ and underestimation of effective population size. Data generated from lower effective population sizes were less likely to fail the date randomization test yet still resulted in substantially upwardly biased estimates of rates, bringing previous estimates of μ from temporally sampled DNA sequences into question. We find that our general criticisms of both the hypothesis of time‐dependent molecular evolution and Bayesian methods to estimate μ from temporally sampled DNA sequences are further reinforced.     

Bergerac-SH3: "frustation" induced by stabilizing the folding nucleus

The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha-spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour.     

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.