MALDI-TOF MS for the identification of cultivable organic-degrading bacteria in contaminated groundwater near unconventional natural gas extraction sites

Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.info:eu-repo/semantics/publishedVersio

Molecular neuropathology of the synapse in sheep with CLN5 Batten disease

© 2015 Published by Wiley Periodicals, Inc. Aims: Synapses represent a major pathological target across a broad range of neurodegenerative conditions. Recent studies addressing molecular mechanisms regulating synaptic vulnerability and degeneration have relied heavily on invertebrate and mouse models. Whether similar molecular neuropathological changes underpin synaptic breakdown in large animal models and in human patients with neurodegenerative disease remains unclear. We therefore investigated whether molecular regulators of synaptic pathophysiology, previously identified in Drosophila and mouse models, are similarly present and modified in the brain of sheep with CLN5 Batten disease. Methods: Gross neuropathological analysis of CLN5 Batten disease sheep and controls was used alongside postmortem MRI imaging to identify affected brain regions. Synaptosome preparations were then generated and quantitative fluorescent Western blotting used to determine and compare levels of synaptic proteins. Results: The cortex was particularly affected by regional neurodegeneration and synaptic loss in CLN5 sheep, whilst the cerebellum was relatively spared. Quantitative assessment of the protein content of synaptosome preparations revealed significant changes in levels of seven out of eight synaptic neurodegeneration proteins investigated in the motor cortex, but not cerebellum, of CLN5 sheep (α-synuclein, CSP-α, neurofascin, ROCK2, calretinin, SIRT2, and UBR4). Conclusions: Synaptic pathology is a robust correlate of region-specific neurodegeneration in the brain of CLN5 sheep, driven by molecular pathways similar to those reported in Drosophila and rodent models. Thus, large animal models, such as sheep, represent ideal translational systems to develop and test therapeutics aimed at delaying or halting synaptic pathology for a range of human neurodegenerative conditions

Cannabidiol and cannabigerol exert antimicrobial activity without compromising skin microbiota

Cannabidiol (CBD) and cannabigerol (CBG) are two pharmacologically active phytocannabinoids of Cannabis sativa L. Their antimicrobial activity needs further elucidation, particularly for CBG, as reports on this cannabinoid are scarce. We investigated CBD and CBG’s antimicrobial potential, including their ability to inhibit the formation and cause the removal of biofilms. Our results demonstrate that both molecules present activity against planktonic bacteria and biofilms, with both cannabinoids removing mature biofilms at concentrations below the determined minimum inhibitory concentrations. We report for the first time minimum inhibitory and lethal concentrations for Pseudomonas aeruginosa and Escherichia coli (ranging from 400 to 3180 µM), as well as the ability of cannabinoids to inhibit Staphylococci adhesion to keratinocytes, with CBG demonstrating higher activity than CBD. The value of these molecules as preservative ingredients for cosmetics was also assayed, with CBG meeting the USP 51 challenge test criteria for antimicrobial effectiveness. Further, the exact formulation showed no negative impact on skin microbiota. Our results suggest that phytocannabinoids can be promising topical antimicrobial agents when searching for novel therapeutic candidates for different skin conditions. Additional research is needed to clarify phytocannabinoids’ mechanisms of action, aiming to develop practical applications in dermatological use.info:eu-repo/semantics/publishedVersio

Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity

The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD

Uncovering memory-related gene expression in contextual fear conditioning using ribosome profiling

Contextual fear conditioning (CFC) in rodents is the most widely used behavioural paradigm in neuroscience research to elucidate the neurobiological mechanisms underlying learning and memory. It is based on the pairing of an aversive unconditioned stimulus (US; e.g. mild footshock) with a neutral conditioned stimulus (CS; e.g. context of the test chamber) in order to acquire associative long-term memory (LTM), which persists for days and even months. Using genome-wide analysis, several studies have generated lists of genes modulated in response to CFC in an attempt to identify the "memory genes", which orchestrate memory formation. Yet, most studies use naïve animals as a baseline for assessing gene-expression changes, while only few studies have examined the effect of the US alone, without pairing to context, using genome-wide analysis of gene-expression. Herein, using the ribosome profiling methodology, we show that in male mice an immediate shock, which does not lead to LTM formation, elicits pervasive translational and transcriptional changes in the expression of Immediate Early Genes (IEGs) in dorsal hippocampus (such as Fos and Arc), a fact which has been disregarded by the majority of CFC studies. By removing the effect of the immediate shock, we identify and validate a new set of genes, which are translationally and transcriptionally responsive to the association of context-to-footshock in CFC, and thus constitute salient "memory genes"

Influence of EPICardial adipose tissue in HEART diseases (EPICHEART) study: Protocol for a translational study in coronary atherosclerosis

Accumulation of epicardial adipose tissue (EAT) is associated with coronary artery disease (CAD) and increased risk of coronary events in asymptomatic subjects and low-risk patients, suggesting that EAT promotes atherosclerosis in its early stage. Recent studies have shown that the presence of CAD affects the properties of adjacent EAT, leading to dynamic changes in the molecular players involved in the interplay between EAT and the coronary arteries over the history of the disease. The role of EAT in late-stage CAD has not been investigated.coronárioResumoIntroduc¸ão:Acumulac¸ão de tecido adiposo epicárdico (TAE) tem sido associado a doenc¸acoronária aterosclerótica (DC) e aumento do risco de eventos coronários em indivíduos ass-intomáticos e doentes de baixo risco, sugerindo que o TAE pode promover fases precoces daDC. Estudo recentes mostraram que a presenc¸a de DC afeta as características do TAE adja-cente levando a modificac¸ões dinâmicas nos mediadores envolvidos na comunicac¸ão entre oTAE e as artérias coronárias ao longo da história da DC. O papel doTAE nas fases avanc¸adas daaterosclerose coronária não foi investigado.Objetivos: Através de análise comparativa com o tecido adiposo mediastínico e subcutâneo,pretendemos investigar se o volume do TAE, avaliado por tomografia computadorizada (TC), eo seu proteoma, avaliado por espectrometria de massa técnica de SWATH, estão associados aestadios avanc¸ados da DC numa coorte de estenose aórtica grave.Métodos: O estudo EPICHEART (NCT03280433) é um estudo prospetivo que inclui doentescom estenose aórtica grave referenciados para substituic¸ão eletiva da válvula aórtica, cujoprotocolo envolve avaliac¸ão pré-operatória clínica, nutricional, ecocardiográfica, por TC eangiografia coronária invasiva. Durante a cirurgia cardíaca, colhemos amostras de tecido adi-poso epicárdico, mediastínico e subcutâneo para análise do seu proteoma por espectrometriade massa técnica de SWATH. Adicionalmente, colhemos líquido pericárdico, sangue venoso per-iférico e do seio coronário para investigar mediadores de DC derivados do TAE na circulac¸ãosistémica e local.Conclusão: Desenhámos um estudo de translac¸ão para explorar a associac¸ão da quantidade equalidade do TAE com a DC tardia. Esperamos identificar mediadores da comunicac¸ão recíprocaentre o TAE e as artérias coronárias que estão envolvidos na patogénese das fases avanc¸adas daDC, especialmente, calcificac¸ão coronária, os quais podem servir como novos alvos terapêuticose soluc¸ões de engenharia biomédica para visualizac¸ão da DC

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species