Predicción del punto de fusión de indoles con base en la estructura molecular usando redes neuronales artificiales

Mediante la aplicación del método de relación cuantitativa de estructura propiedad se determinó un modelo para predecir la temperatura del punto de fusión de indoles a partir de su estructura molecular (n = 86). Usando los programas de computadora Gaussian 98 y PCDM 2.0, se calcularon una serie de descriptores moleculares; descriptores electrónicos, topológicos y geométricos. Para la elaboración del modelo de predicción se empleó la regresiónlinear múltiple entre los descriptores moleculares y la temperatura de los puntos de fusión de los indoles presentes en la base de datos. Dando como resultados un coeficiente de determinación (R2) y un error estándar de estimación (EEE) de 0.73 y 27.42°C respectivamente. Por medio de una red neuronal retropropagación (5: 4: 1) se optimizó el modelo de regresión lineal múltiple, pudiéndose incluir relaciones no lineales entre la estructura molecular y la temperatura del punto de fusión de los indoles, obteniendo mejores resultados en la predicción del puntode fusión para el grupo de entrenamiento (R2 =0.9978) y el grupo de validación (R2 =0.9987). El error cuadrático promedio (MSE) asociado al grupo de entrenamiento y de validación para el modelo de con la red fue 0.006 y 0.006 respectivamente

Expression, Characterization and Synergistic Interactions of Myxobacter Sp. AL-1 Cel9 and Cel48 Glycosyl Hydrolases

The soil microorganism Myxobacter Sp. AL-1 regulates in a differential manner the production of five extracellular cellulases during its life cycle. The nucleotide sequence of a cel9-cel48 cluster from the genome of this microorganism was recently obtained. Cel48 was expressed in Escherichia coli to generate a His6-Cel48 protein and the biochemical properties of the pure protein were determined. Cel48 was more efficient in degrading acid-swollen avicel (ASC) than carboxymethylcellulose (CMC). On the other hand, cel9 was expressed in Bacillus subtilis from an IPTG-inducible promoter. Zymogram analysis showed that after IPTG-induction, Cel9 existed in both the cell fraction and the culture medium of B. subtilis and the secreted protein was purified to homogeneity by FPLC-ionic exchange chromatography. The exocellobiohydrolase Cel48 showed a synergism of 1.68 times with the endocellulase Cel9 during ASC degradation using an 8.1-fold excess of Cel48 over Cel9. Western blot analysis revealed that both proteins were synthesized and secreted to the culture medium of Myxobacter Sp. AL-1. These results show that the cel9-cel48 cluster encodes functional endo- and exo-acting cellulases that allows Myobacter Sp. AL-1 to hydrolyse cellulose

The functional differences between paralogous regulators define the control of the general stress response in Sphingopyxis granuli TFA

Copyright © 2022 The Authors. Sphingopyxis granuli TFA is a contaminant degrading alphaproteobacterium that responds to adverse conditions by inducing the general stress response (GSR), an adaptive response that controls the transcription of a variety of genes to overcome adverse conditions. The core GSR regulators (the response regulator PhyR, the anti-σ factor NepR and the σ factor EcfG) are duplicated in TFA, being PhyR1 and PhyR2, NepR1 and NepR2 and EcfG1 and EcfG2. Based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each paralogue and assessed their contribution to the GSR regulation, dictating its timing and the intensity. We show that different stress signals are differentially integrated into the GSR by PhyR1 and PhyR2, therefore producing different levels of GSR activation. We demonstrate in vitro that both NepR1 and NepR2 bind EcfG1 and EcfG2, although NepR1 produces a more stable interaction than NepR2. Conversely, NepR2 interacts with phosphorylated PhyR1 and PhyR2 more efficiently than NepR1. We propose an integrative model where NepR2 would play a dual negative role: it would directly inhibit the σ factors upon activation of the GSR and it would modulate the GSR activity indirectly by titrating the PhyR regulators.Ministerio de Educación y Ciencia. Grant Number: FPU15/04789; Spanish Ministry for Education and Science and the European Regional Development Fund. Grant Number: BIO2014-57545-R

Extracytoplasmic Function σ Factors as Tools for Coordinating Stress Responses

Copyright: © 2021 by the authors. The ability of bacterial core RNA polymerase (RNAP) to interact with different σ factors, thereby forming a variety of holoenzymes with different specificities, represents a powerful tool to coordinately reprogram gene expression. Extracytoplasmic function σ factors (ECFs), which are the largest and most diverse family of alternative σ factors, frequently participate in stress responses. The classification of ECFs in 157 different groups according to their phylogenetic relationships and genomic context has revealed their diversity. Here, we have clustered 55 ECF groups with experimentally studied representatives into two broad classes of stress responses. The remaining 102 groups still lack any mechanistic or functional insight, representing a myriad of systems yet to explore. In this work, we review the main features of ECFs and discuss the different mechanisms controlling their production and activity, and how they lead to a functional stress response. Finally, we focus in more detail on two well-characterized ECFs, for which the mechanisms to detect and respond to stress are complex and completely different: Escherichia coli RpoE, which is the best characterized ECF and whose structural and functional studies have provided key insights into the transcription initiation by ECF-RNAP holoenzymes, and the ECF15-type EcfG, the master regulator of the general stress response in AlphaproteobacteriaBIO2014-57545-R and PGC2018-097151-B-I00, cofunded by the Spanish Ministerio de Ciencia e Innovación and the European Regional Development Fund; fellowship from the FPU program (Ministerio de Universidades, Spain)

Opioid pain medication prescription for chronic pain in primary care centers: the roles of pain acceptance, pain intensity, depressive symptoms, pain catastrophizing, sex, and age

Background: Psychological factors of patients may influence physicians’ decisions on prescribing opioid analgesics. However, few studies have sought to identify these factors. The present study had a double objective: (1) To identify the individual factors that differentiate patients who had been prescribed opioids for the management of chronic back pain from those who had not been prescribed opioids and (2) to determine which factors make significant and independent contributions to the prediction of opioid prescribing. Methods: A total of 675 patients from four primary care centers were included in the sample. Variables included sex, age, pain intensity, depressive symptoms, pain catastrophizing, and pain acceptance. Results: Although no differences were found between men and women, participants with chronic noncancer pain who were prescribed opioids were older, reported higher levels of pain intensity and depressive symptoms, and reported lower levels of pain-acceptance. An independent association was found between pain intensity and depressive symptoms and opioid prescribing. Conclusions: The findings suggest that patient factors influence physicians’ decisions on prescribing opioids. It may be useful for primary care physicians to be aware of the potential of these factors to bias their treatment decisions.info:eu-repo/semantics/publishedVersio

An improved genome editing system for Sphingomonadaceae

This is a preprint, which is a preliminary version of an article and has not completed peer review.The sphingomonads encompass a diverse group of bacteria within the Sphingomonadaceae family, with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their capability to degrade or metabolise a variety of recalcitrant organic pollutants. However, the research and development of their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for Sphingopyxis granuli, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious re-streaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting ecfG2, the master regulator of the general stress response in S. granuli. This adds onto the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.This study was supported by the: Spanish National Plan for Scientific and Technical Research and Innovation (Award PID2021-125491NB-I00), Principle Award Recipient: Francisca Reyes-Ramírez; Junta de Andalucía (Award POSTDOC_21_00064), Principle Award Recipient: Inmaculada García-Romero; Biotechnology and Biological Sciences Research Council (Award BB/V007823/1), Principle Award Recipient: Not Applicable

Two paralogous EcfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli TFA

© The Author(s) 2020. Under ever-changing environmental conditions, the General Stress Response (GSR) represents a lifesaver for bacteria in order to withstand hostile situations. In α-proteobacteria, the EcfG-type extracytoplasmic function (ECF) σ factors are the key activators of this response at the transcriptional level. In this work, we address the hierarchical function of the ECF σ factor paralogs EcfG1 and EcfG2 in triggering the GSR in Sphingopyxis granuli TFA and describe the role of EcfG2 as global switch of this response. In addition, we define a GSR regulon for TFA and use in vitro transcription analysis to study the relative contribution of each EcfG paralog to the expression of selected genes. We show that the features of each promoter ultimately dictate this contribution, though EcfG2 always produced more transcripts than EcfG1 regardless of the promoter. These first steps in the characterisation of the GSR in TFA suggest a tight regulation to orchestrate an adequate protective response in order to survive in conditions otherwise lethal.Grant BIO2014–57545-R, co-funded by the Spanish Ministerio de Educación y Ciencia and the European Regional Development Fund; fellowships from the FPU program (Ministerio de Educación y Ciencia, Spain)

Identification of two fnr genes and characterisation of their role in the anaerobic switch in Sphingopyxis granuli strain TFA

Copyright © The Author(s) 2020. Sphingopyxis granuli strain TFA is able to grow on the organic solvent tetralin as the only carbon and energy source. The aerobic catabolic pathway for tetralin, the genes involved and their regulation have been fully characterised. Unlike most of the bacteria belonging to the sphingomonads group, this strain is able to grow in anoxic conditions by respiring nitrate, though not nitrite, as the alternative electron acceptor. In this work, two fnr-like genes, fnrN and fixK, have been identified in strain TFA. Both genes are functional in E. coli and Sphingopyxis granuli although fixK, whose expression is apparently activated by FnrN, seems to be much less effective than fnrN in supporting anaerobic growth. Global transcriptomic analysis of a ΔfnrN ΔfixK double mutant and identification of Fnr boxes have defined a minimal Fnr regulon in this bacterium. However, expression of a substantial number of anaerobically regulated genes was not affected in the double mutant. Additional regulators such regBA, whose expression is also activated by Fnr, might also be involved in the anaerobic response. Anaerobically induced stress response genes were not regulated by Fnr but apparently induced by stress conditions inherent to anaerobic growth, probably due to accumulation of nitrite and nitric oxide.Grants BIO2014-57545-R and PGC2018-097151-B-I00, co-funded by the Spanish Ministerio de Ciencia e Innovación and the European Regional Development Fund; fellowships from the FPU program (Ministerio de Universidades, Spain)