Linking Pedobacter lusitanus NL19 volatile exometabolome with growth medium composition: what can we learn using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry?

Microbial metabolomics allows understanding and to comprehensively analyse metabolites, and their related cellular and metabolic processes, that are produced and released to the extracellular environment under specific conditions. In that regard, the main objective of this research is to understand the impact of culture media changes in the metabolic profile of Pedobacter lusitanus NL19 (NL19) and Pedobacter himalayensis MTCC 6384 (MTCC6384) and respective influence on the production of biotechnologically relevant compounds. Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry with time-of-flight analyser (GC × GC-ToFMS) was applied to comprehensively study the metabolites produced by NL19 and MTCC6384 both in tryptic soy broth 100% (TSB100) and tryptic soy broth with 25% casein peptone (PC25). A total of 320 metabolites were putatively identified, which belong to different chemical families: alcohols, aldehydes, esters, ethers, hydrocarbons, ketones, nitrogen compounds, sulphur compounds, monoterpenes, and sesquiterpenes. Metabolites that were statistically different from the control (sterile medium) were selected allowing for the construction of the metabolic profile of both strains. A set of 80 metabolites was tentatively associated to the metabolic pathways such as the metabolism of fatty acids, branched-chain aminoacids, phenylalanine, methionine, aromatic compounds, and monoterpene and sesquiterpene biosynthesis. This study allowed to better understand how slight changes of the culture media and thus the composition of nutrients impair the metabolic profile of bacteria, which may be further explored for metabolomics pipeline construction or biotechnological applications.

Pharmacological Signature and Target Specificity of Inhibitory Circuits Formed by Martinotti Cells in the Mouse Barrel Cortex.

In the neocortex, fast synaptic inhibition orchestrates both spontaneous and sensory-evoked activity. GABAergic interneurons (INs) inhibit pyramidal neurons (PNs) directly, modulating their output activity and thus contributing to balance cortical networks. Moreover, several IN subtypes also inhibit other INs, forming specific disinhibitory circuits, which play crucial roles in several cognitive functions. Here, we studied a subpopulation of somatostatin-positive INs, the Martinotti cells (MCs) in layer 2/3 of the mouse barrel cortex (both sexes). MCs inhibit the distal portion of PN apical dendrites, thus controlling dendrite electrogenesis and synaptic integration. Yet, it is poorly understood whether MCs inhibit other elements of the cortical circuits, and the connectivity properties with non-PN targets are unknown. We found that MCs have a strong preference for PN dendrites, but they also considerably connect with parvalbumin-positive, vasoactive intestinal peptide-expressing, and layer 1 (L1) INs. Remarkably, GABAergic synapses from MCs exhibited clear cell type-specific short-term plasticity. Moreover, whereas the biophysical properties of MC-PN synapses were consistent with distal dendritic inhibition, MC-IN synapses exhibited characteristics of fast perisomatic inhibition. Finally, MC-PN connections used α5-containing GABAA receptors (GABAARs), but this subunit was not expressed by the other INs targeted by MCs. We reveal a specialized connectivity blueprint of MCs within different elements of superficial cortical layers. In addition, our results identify α5-GABAARs as the molecular fingerprint of MC-PN dendritic inhibition. This is of critical importance, given the role of α5-GABAARs in cognitive performance and their involvement in several brain diseases.SIGNIFICANCE STATEMENT Martinotti cells (MCs) are a prominent, broad subclass of somatostatin-expressing GABAergic interneurons, specialized in controlling distal dendrites of pyramidal neurons (PNs) and taking part in several cognitive functions. Here we characterize the connectivity pattern of MCs with other interneurons in the superficial layers (L1 and L2/3) of the mouse barrel cortex. We found that the connectivity pattern of MCs with PNs as well as parvalbumin, vasoactive intestinal peptide, and L1 interneurons exhibit target-specific plasticity and biophysical properties. The specificity of α5-GABAARs at MC-PN synapses and the lack or functional expression of this subunit by other cell types define the molecular identity of MC-PN connections and the exclusive involvement of this inhibitory circuits in α5-dependent cognitive tasks.

Assessing the potential of the two-peptide lantibiotic lichenicidin as a new generation antimicrobial.

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliß. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliß. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative.

An approach to assess and identify polymers in the health-care waste of a Brazilian university hospital.

This work presents the health-care waste (HCW) management and an approach to assess and identify polymers in a General Surgery Unit - Internment Service (GSU) of a Brazilian university hospital, to estimate the main polymers presenting in medical devices that are consumed during a year, discarded either as infecting (Group A) or as scarifying residue (Group E). Among the waste produced from the medical devices, 3.14 ton (98.79%) were composed of polymers (63.06% of plastics and 35.73% elastomers) while around 0.03 ton (1.21%) by metals. The proposed approach is composed of 4 steps: (1) Collecting data about consumed medical devices to be categorized into the residues Groups (A and E); (2) Identifying the polymeric composition with information provided by suppliers; (3) Characterizing the polymer functional groups by Fourier-Transform Infrared Spectroscopy (FTIR) and (4) Determining the polymer melting point by Differential Scanning Calorimetry (DSC). According to the results, the analyzed HCW was composed mainly of polypropylene (80.88%), high-density polyethylene (5.28%), polystyrene (4.51%), and cellulose (3.58%), from a total of 11 different polymers.

SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine.

The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.

Transcript profiling and DNA damage in the European eel (Anguilla anguilla L.) exposed to 7,12-dimethylbenz[a]anthracene.

The molecular responses induced during and after an acute exposure to 7,12-dimethylbenz[a]anthracene (DMBA) were analysed in liver, gill and blood cells of juvenile Anguilla anguilla with the aim of developing molecular biomarkers of environmental PAH pollution. Changes in the mRNA expression levels of the cell cycle checkpoint-related rad1 gene and the mRNAs of differentially expressed genes by suppression subtractive hybridization (SSH) were analysed in the liver, and related to well-established biomarkers: cyp1A1 mRNA expression and assessment of the DNA integrity using the comet assay and flow cytometry. DMBA exposure resulted in increased cyp1A1 mRNA levels, suggesting that cyp1A1 might be involved in the metabolism of DMBA. Global DNA damage, detected by the comet assay, was observed in the three tissues analysed but only blood cells showed chromosomal lesions as analysed by flow cytometry. Although DNA damage was found in the liver, no induction in rad1 gene was observed in this organ. The global SSH approach revealed that mRNAs of genes related to xenobiotic metabolism, immune processes and cytoskeleton dynamics were differentially expressed in DMBA-exposed eel livers, highlighting the complexity in the response observed in fish exposed to a genotoxic agent and providing directions for new biomarker development.

Mutation analysis of ras gene in the liver of European eel (Anguilla anguilla L.) exposed to benzo[a]pyrene.

Ras is regarded as one of the most important genes involved in carcinogenesis. Such genes have been characterised in several fish species and the presence of ras mutations have already been described in fish populations from hydrocarbon contaminated areas and following experimental exposure to specific contaminants. The aims of this study were to evaluate the DNA integrity by comet assay, to isolate the normal ras gene of Anguilla anguilla and analyse for the presence of ras gene mutations or changes in gene expression levels induced after one month of benzo[a]pyrene (BaP) experimental exposure. The A. anguilla ras cDNA isolated revealed a 189 amino acid protein and alignment with other vertebrate ras proteins revealed conservation of functionally important regions. Following experimental exposure to BaP, an increase in DNA damage was found by comet assay. However, no point mutations or changes in ras gene expression levels were detected when compared to control samples. In contrast to the majority of fish ras gene sequences, a high degree of polymorphic variation was detected in the A. anguilla ras gene.