Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Distinguishing colour variants of <i>Serapias perez-chiscanoi</i> (Orchidaceae) from related taxa on the Iberian Peninsula

Serapias perez-chiscanoi has a stable and uniform appearance with green flowers. Throughout its distribution area, however, plants have been found with deviant pink to red flowers that show similarities with other taxa that are occasionally pale flowered. S. perez-chiscanoi is easy to differentiate from S. cordigera subsp. cordigera by the colour of the flowers (S. cordigera subsp. cordigera has red to purple flowers) and the fact that the hypochile dimensions of S. perez-chiscanoi are significantly smaller. It is, however, more difficult to distinguish it from individuals of S. cordigera subsp. gentilii with pale flowers, which occur frequently. The two taxa differ in colour pattern and floral dimensions, especially the hypochile length, which is shorter in S. perez-chiscanoi. Pale-flowered individuals of another species, S. parviflora, are easily distinguished by their significantly smaller flowers. S. perez-chiscanoi occurs in Spain in the autonomous regions of Extremadura and Castilla-La Mancha and in Portugal, S. cordigera subsp. gentilii seems to occur along the coastal regions of SW Portugal, while S. cordigera subsp. cordigera and S. parviflora are distributed throughout the Iberian Peninsula

A novel non-peptidic agonist of the ghrelin receptor with orexigenic activity in vivo

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand

The Deleterious Influence of Tenofovir-Based Therapies on the Progression of Atherosclerosis in HIV-Infected Patients

We investigated the potential differential effects of antiretroviral therapies on unbalanced chemokine homeostasis and on the progression of atherosclerosis in HIV-infected patients. A two-year prospective study was performed in 67 consecutive HIV-infected patients initiating antiretroviral therapy with abacavir/lamivudine or tenofovir/emtricitabine. Circulating levels of inflammatory biomarkers, progression of subclinical atherosclerosis and expression levels of selected chemokines genes in circulating leukocytes were assessed. Control subjects showed significantly lower plasma concentrations of CRP, tPA, IL-6, and MCP-1 than HIV-infected patients at a baseline. After two years of followup, the observed decreases in plasma inflammatory biomarker levels were only significant for MCP-1, tPA, and IL-6. The decrease in plasma MCP-1 concentration was associated with the progression of atherosclerosis, and this effect was negligible only in patients receiving TDF-based therapy. Multivariate analysis confirmed that treatment with TDF was positively and significantly associated with a higher likelihood of subclinical atherosclerosis progression. However, the expression levels of selected genes in blood cells only showed associations with the viral load and total and HDL-cholesterol levels. Current antiretroviral treatments may partially attenuate the influence of HIV infection on certain inflammatory pathways, though patients receiving TDF therapy must be carefully monitored with respect to the presence and/or progression of atherosclerosis

Access to Some C5-Cyclised 2-Pyrones and 2-Pyridones via Direct Arylation; Retention of Chloride as a Synthetic Handle

The synthetic effort towards the functionalisation of C–H bonds on 2-pyrones and 2-pyridones has been enabled by the preferential reactivity of the C-3 position. Herein, we report a direct arylation protocol for the intramolecular coupling of 2-pyrones and 2-pyridones, allowing access to a previously unavailable class of C-5 cyclised products with an unstudied biological profile. A C–Cl bond was retained at C-3 during the direct arylation process allowing further derivatisation at C-3, using a Suzuki–Miyaura cross-coupling reaction

MiRNA Expression Profile of Human Subcutaneous Adipose and during Adipocyte Differentiation

BACKGROUND: Potential regulators of adipogenesis include microRNAs (miRNAs), small non-coding RNAs that have been recently shown related to adiposity and differentially expressed in fat depots. However, to date no study is available, to our knowledge, regarding miRNAs expression profile during human adipogenesis. Thereby, the aim of this study was to investigate whether miRNA pattern in human fat cells and subcutaneous adipose tissue is associated to obesity and co-morbidities and whether miRNA expression profile in adipocytes is linked to adipogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We performed a global miRNA expression microarray of 723 human and 76 viral mature miRNAs in human adipocytes during differentiation and in subcutaneous fat samples from non-obese (n = 6) and obese with (n = 9) and without (n = 13) Type-2 Diabetes Mellitus (DM-2) women. Changes in adipogenesis-related miRNAs were then validated by RT-PCR. Fifty of 799 miRNAs (6.2%) significantly differed between fat cells from lean and obese subjects. Seventy miRNAs (8.8%) were highly and significantly up or down-regulated in mature adipocytes as compared to pre-adipocytes. Otherwise, 17 of these 799 miRNAs (2.1%) were correlated with anthropometrical (BMI) and/or metabolic (fasting glucose and/or triglycerides) parameters. We identified 11 miRNAs (1.4%) significantly deregulated in subcutaneous fat from obese subjects with and without DM-2. Interestingly, most of these changes were associated with miRNAs also significantly deregulated during adipocyte differentiation. CONCLUSIONS/SIGNIFICANCE: The remarkable inverse miRNA profile revealed for human pre-adipocytes and mature adipocytes hints at a closely crosstalk between miRNAs and adipogenesis. Such candidates may represent biomarkers and therapeutic targets for obesity and obesity-related complications

Antibiotics and Antimicrobial Resistance in the COVID-19 Era: Perspective from Resource-Limited Settings

The dissemination of COVID-19 around the globe has been followed by an increased consumption of antibiotics. This is related to the concern for bacterial superinfection in COVID-19 patients. The identification of bacterial pathogens is challenging in low and middle income countries (LMIC), as there are no readily-available and cost-effective clinical or biological markers that can effectively discriminate between bacterial and viral infections. Fortunately, faced with the threat of COVID-19 spread, there has been a growing awareness of the importance of antimicrobial stewardship programs, as well as infection prevention and control measures that could help reduce the microbial load and hence circulation of pathogens, with a reduction in dissemination of antimicrobial resistance. These measures should be improved particularly in developing countries. Studies need to be conducted to evaluate the worldwide evolution of antimicrobial resistance during the COVID-19 pandemic, because pathogens do not respect borders. This issue takes on even greater importance in developing countries, where data on resistance patterns are scarce, conditions for infectious pathogen transmission are optimal, and treatment resources are suboptimal

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a ligninrelated phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.This work was funded by INDOX (KBBE-2013-7-613549) European project and NOESIS (BIO2014-56388-R) and CTQ2013- 48287-R Spanish National Projects. I. P. and G. S. acknowledge the Spanish Research Council (CSIC) and MINECO for their respective predoctoral fellowships.Peer ReviewedPostprint (published version

Initiation and continuation of randomized trials after the publication of a trial stopped early for benefit asking the same study question: STOPIT-3 study design

Abstract Background: Randomized control trials (RCTs) stopped early for benefit (truncated RCTs) are increasingly common and, on average, overestimate the relative magnitude of benefit by approximately 30%. Investigators stop trials early when they consider it is no longer ethical to enroll patients in a control group. The goal of this systematic review is to determine how investigators of ongoing or planned RCTs respond to the publication of a truncated RCT addressing a similar question. Methods/design: We will conduct systematic reviews to update the searches of 210 truncated RCTs to identify similar trials ongoing at the time of publication, or started subsequently, to the truncated trials (&apos;subsequent RCTs&apos;). Reviewers will determine in duplicate the similarity between the truncated and subsequent trials. We will analyze the epidemiology, distribution, and predictors of subsequent RCTs. We will also contact authors of subsequent trials to determine reasons for beginning, continuing, or prematurely discontinuing their own trials, and the extent to which they rely on the estimates from truncated trials. Discussion: To the extent that investigators begin or continue subsequent trials they implicitly disagree with the decision to stop the truncated RCT because of an ethical mandate to administer the experimental treatment. The results of this study will help guide future decisions about when to stop RCTs early for benefit