Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels.

The chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl- efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl- efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl- efflux.

Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

Abdominal normothermic regional perfusion in controlled donation after circulatory determination of death liver transplantation: Outcomes and risk factors for graft loss.

Postmortem normothermic regional perfusion (NRP) is a rising preservation strategy in controlled donation after circulatory determination of death (cDCD). Herein, we present results for cDCD liver transplants performed in Spain 2012-2019, with outcomes evaluated through December 31, 2020. Results were analyzed retrospectively and according to recovery technique (abdominal NRP [A-NRP] or standard rapid recovery [SRR]). During the study period, 545 cDCD liver transplants were performed with A-NRP and 258 with SRR. Median donor age was 59 years (interquartile range 49-67 years). Adjusted risk estimates were improved with A-NRP for overall biliary complications (OR 0.300, 95% CI 0.197-0.459, p < .001), ischemic type biliary lesions (OR 0.112, 95% CI 0.042-0.299, p < .001), graft loss (HR 0.371, 95% CI 0.267-0.516, p < .001), and patient death (HR 0.540, 95% CI 0.373-0.781, p = .001). Cold ischemia time (HR 1.004, 95% CI 1.001-1.007, p = .021) and re-transplantation indication (HR 9.552, 95% CI 3.519-25.930, p < .001) were significant independent predictors for graft loss among cDCD livers with A-NRP. While use of A-NRP helps overcome traditional limitations in cDCD liver transplantation, opportunity for improvement remains for cases with prolonged cold ischemia and/or technically complex recipients, indicating a potential role for complimentary ex situ perfusion preservation techniques.

Leadership, management, quality, and innovation in organ donation: 2019 Kunming recommendations for One Belt & One Road countries.

The findings and recommendations of the 2019 consensus conference in organ donation, held in Kunming, China, are here reported. The main objective of the conference was to gather relevant information from experts involved in the field. The data and opinions provided allowed to propose a series of recommendations for "One Belt & One Road Countries" on how to achieve self-sufficiency in organ donation. Leadership in organ donation should be results-oriented and goal-driven based on the principles of excellence, empowerment, and engagement, providing the means, resources, and strategies necessary to reach the goal in earnest. Management includes good governance and transparency of a national registry of patients in the waiting list, donors, transplants, transplant teams, quality, and safety programs with continuous educational training of health care professionals. Mandatory monitoring, auditing and evaluation of quality must be incorporated into donation practices as relevant points in innovation, as well as the adoption of already established and novel processes and technologies. Achievement of self-sufficiency in organ donation is a crucial step to fight against transplant tourism and to prevent organ trafficking. Based on recommendations arising from the conference, each country could review and develop individualized action plans adjusted to its own circumstances and reality.

Discovery of Diverse Natural Products as Inhibitors of SARS-CoV-2 Mpro Protease through Virtual Screening.

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.

Fragment dissolved molecular dynamics: a systematic and efficient method to locate binding sites.

Diverse computational methods to support fragment-based drug discovery (FBDD) are available in the literature. Despite their demonstrated efficacy in supporting FBDD campaigns, they exhibit some drawbacks such as protein denaturation or ligand aggregation that have not yet been clearly overcome in the framework of biomolecular simulations. In the present work, we discuss a systematic semi-automatic novel computational procedure, designed to surpass these difficulties. The method, named fragment dissolved Molecular Dynamics (fdMD), utilizes simulation boxes of solvated small fragments, adding a repulsive Lennard-Jones potential term to avoid aggregation, which can be easily used to solvate the targets of interest. This method has the advantage of solvating the target with a low number of ligands, thus preventing the denaturation of the target, while simultaneously generating a database of ligand-solvated boxes that can be used in further studies. A number of scripts are made available to analyze the results and obtain the descriptors proposed as a means to trustfully discard spurious binding sites. To test our method, four test cases of different complexity have been solvated with ligand boxes and four molecular dynamics runs of 200 ns length have been run for each system, which have been extended up to 1 µs when needed. The reported results point out that the selected number of replicas are enough to identify the correct binding sites irrespective of the initial structure, even in the case of proteins having several close binding sites for the same ligand. We also propose a set of descriptors to analyze the results, among which the average MMGBSA and the average KDEEP energies have emerged as the most robust ones.

Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome's "health" and/or subsequently resolve Blastocystis' potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

Chronic viral hepatitis C micro-elimination program using telemedicine. The Mexican experience.

BACKGROUND: hepatitis C virus (HCV) infection is a global health problem. Chronic infection induces the development of fibrosis and cirrhosis together with all the related complications. The use of direct-acting antiviral (DAA) drugs has proven highly effective. Telemedicine is a present-day resource that brings treatment closer to distant areas and may result in cost savings. OBJECTIVE: to implement a microelimination program for HCV using DAAs with the support of a telemedicine program to minimize expenses. PATIENTS AND METHODS: the program was developed at the Medical Services department of Petróleos Mexicanos (SMPM) with a national coverage; patients diagnosed with chronic hepatitis C were included. These were classified into locals and outsiders. Treatment for foreign patients was indicated, monitored and completed via telemedicine. Thus, avoiding their travel to the country's capital city, in order to save on transportation costs and travel allowances. RESULTS: a total of 136 patients, 74 locals and 62 outsiders, participated in the study. Transfer was avoided for 62 patients (45.5 %), which meant that telemedicine resulted in savings of 3,176.20 USD per patient, with overall savings of 196,924.40 USD from cost minimization. A total of 30 patients remained untreated due to lack of medication, hence the coverage amounted to 86 %. Sustained virological response (SVR) was achieved in 99 % of cases. Only two patients had treatment failure. Adverse events included headache and fatigue in 5 % of the cohort. CONCLUSIONS: with the aid of a telemedicine approach, significant savings were achieved by minimizing costs, since nearly half of patients were outsiders. Coverage reached 86 % and treatment with DAAs was successful for 99 % of our cases.

Molecular Determinants for the Activation/Inhibition of Bak Protein by BH3 Peptides.

Apoptosis is a key cell death pathway in mammalian cells. Understanding this process and its regulation has been a subject of study in the last three decades. Members of the Bcl-2 family of proteins are involved in the regulation of apoptosis through mitochondrial poration with the subsequent initiation of apoptosis. Deregulation of proapoptotic proteins contributes to the progression of many tumor processes. Understanding how these pore-forming Bcl-2 proteins Bak and Bax are activated is key to find new anticancer treatments. As no drug capable of activating Bak has been disclosed yet, the study of the structural features of BH3 peptides-known as Bak activators-relevant for binding along with its binding energy decomposition analysis, becomes essential for designing novel small-molecule mimics of BH3. Interestingly, a BH3 Bim analogue-inactivating Bak has recently been discovered, opening a question on the molecular features that determine the functions of BH3 peptides. Therefore, the present work is aimed at understanding the way BH3 peptides activate or inactivate Bak in order to identify differential structural features that can be used in drug design. For this purpose, complexes of Bak with an activator and an inhibitor have been subjected to a molecular dynamics study. Structural differences were assessed by means of the fluctuations of the corresponding principal components. Moreover, the MMPB/GBSA approach was used to compute the binding free energy of the diverse complexes to identify those residues of the BH3 peptide that exhibit the larger contributions to complex formation. The results obtained in this work show differences between activators and inhibitors, both in structural and energetic terms, which can be used in the design of new molecules that can activate or inactivate proapoptotic Bak.

Risk of cardiovascular involvement in pediatric patients with X-linked hypophosphatemia.

OBJECTIVE: To find out if cardiovascular alterations are present in pediatric patients with X-linked hypophosphatemia (XLH). STUDY DESIGN: Multicentre prospective clinical study on pediatric patients included in the RenalTube database ( www.renaltube.com ) with genetically confirmed diagnosis of XLH by mutations in the PHEX gene. The study's protocol consisted of biochemical work-up, 24-h ambulatory blood pressure monitoring (ABPM), carotid ultrasonography, and echocardiogram. All patients were on chronic treatment with phosphate supplements and 1-hydroxy vitamin D metabolites. RESULTS: Twenty-four patients (17 females, from 1 to 17 years of age) were studied. Serum concentrations (X ± SD) of phosphate and intact parathyroid hormone were 2.66 ± 0.60 mg/dl and 58.3 ± 26.8 pg/ml, respectively. Serum fibroblast growth factor 23 (FGF23) concentration was 278.18 ± 294.45 pg/ml (normal < 60 pg/ml). Abnormally high carotid intima media thickness was found in one patient, who was obese and hypertensive as revealed by ABPM, which disclosed arterial hypertension in two other patients. Z scores for echocardiographic interventricular septum end diastole and left ventricular posterior wall end diastole were + 0.77 ± 0.77 and + 0.94 ± 0.86, respectively. Left ventricular mass index (LVMI) was 44.93 ± 19.18 g/m2.7, and four patients, in addition to the obese one, had values greater than 51 g/m2.7, indicative of left ventricular hypertrophy. There was no correlation between these echocardiographic parameters and serum FGF23 concentrations. CONCLUSIONS: XLH pediatric patients receiving conventional treatment have echocardiographic measurements of ventricular mass within normal reference values, but above the mean, and 18% have LVMI suggestive of left ventricular hypertrophy without correlation with serum FGF23 concentrations. This might indicate an increased risk of cardiovascular involvement in XLH.

Inspecting the Electronic Architecture and Semiconducting Properties of a Rosette-Like Supramolecular Columnar Liquid Crystal.

Computational and experimental studies unravel the structural and electronic properties of a novel supramolecular liquid crystal built through a hierarchical assembly process resulting in an H-bonded melamine rosette decorated with peripheral triphenylenes. The six-fold symmetry of the mesogen facilitates the formation of a highly organized hexagonal columnar mesophase stable at room temperature. X-ray diffraction and electron density maps confirm additional intra- and intercolumn segregation of functional subunits, and this paves the way for 1D charge transport. Indeed, hole mobility has been measured and found to be higher than for related mesogens. DFT calculations of HOMO and LUMO levels and parameters such as reorganization energy and transfer integral of the rosette structure have been achieved, and not only validate the columnar organization but also establish the way it translates into a favorable electronic architecture and molecular orbital interactions to promote charge carrier mobility.

Molecular Dynamics Analysis of Charge Transport in Ionic-Liquid Electrolytes Containing Added Salt with Mono, Di, and Trivalent Metal Cations.

Among many other applications, room-temperature ionic liquids (ILs) are used as electrolytes for storage and energy-conversion devices. In this work, we investigate, at the microscopic level, the structural and dynamical properties of 1-methyl-1-butyl-pyrrolidinium bis(trifluoromethanesulfonyl) imide [C4 PYR]+ [Tf2 N]- IL-based electrolytes for metal-ion batteries. We carried out molecular dynamics simulations of electrolytes mainly composed of [C4 PYR]+ [Tf2 N]- IL with the addition of Mn+ -[Tf2 N]- metal salts (M=Li+ , Na+ , Ni2+ , Zn2+ , Co2+ , Cd2+ , and Al3+ , n=1, 2, and 3) dissolved in the IL. The addition of low salt concentrations lowers the charge transport and conductivity of the electrolytes. This effect is due to the strong interaction of the metal cations with the [Tf2 N]- anions, which allows for molecular aggregation between them. We analyze how the conformation of the [Tf2 N]- anions surrounding the metal cations determine the charge-transport properties of the electrolyte. We found two main conformations based on the size and charge of the metal cation: monodentate and bidentate (number of oxygen atoms of the anion pointing to the metal atoms). The microscopic local structure of the Mn+ -[Tf2 N]- aggregates influences the microscopic charge transport as well as the macroscopic conductivity of the total electrolyte.

Fitting electron density as a physically sound basis for the development of interatomic potentials of complex alloys.

The development of new interatomic potentials to model metallic systems is a difficult task, due in part to the dependence between the parameters that describe the electron density and the short-range interactions. Parameter search methods are prone to false convergence. To solve this problem, we have developed a methodology for obtaining the electron density parameters independently of the short-range interactions, so that physically sound parameters can be obtained to describe the electron density, after which the short-range parameters can be fitted, thus reducing the complexity of the process and yielding better interatomic potentials. With the new method we can develop self-consistent, accurate force fields, using solely calculations, without the need to fit to experimental data. Density functional theory calculations are used to compute the observables with which the potential is fit. We applied the method to a Ni-based Inconel 625 superalloy (IN625), modelled here as Ni, Cr, Mo and Fe solid solution alloys. The capability of the force fields developed using this new method is validated, by comparing the structural and thermo-elastic properties predicted with the force fields, with the corresponding experimental data, both for single crystals and polycrystalline alloys.

Optimal Scheduling and Fair Service Policy for STDMA in Underwater Networks with Acoustic Communications.

In this work, a multi-hop string network with a single sink node is analyzed. A periodic optimal scheduling for TDMA operation that considers the characteristic long propagation delay of the underwater acoustic channel is presented. This planning of transmissions is obtained with the help of a new geometrical method based on a 2D lattice in the space-time domain. In order to evaluate the performance of this optimal scheduling, two service policies have been compared: FIFO and Round-Robin. Simulation results, including achievable throughput, packet delay, and queue length, are shown. The network fairness has also been quantified with the Gini index.

A Review of the Human Sigma-1 Receptor Structure.

The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.

Objective Video Quality Assessment Based on Machine Learning for Underwater Scientific Applications.

Video services are meant to be a fundamental tool in the development of oceanic research. The current technology for underwater networks (UWNs) imposes strong constraints in the transmission capacity since only a severely limited bitrate is available. However, previous studies have shown that the quality of experience (QoE) is enough for ocean scientists to consider the service useful, although the perceived quality can change significantly for small ranges of variation of video parameters. In this context, objective video quality assessment (VQA) methods become essential in network planning and real time quality adaptation fields. This paper presents two specialized models for objective VQA, designed to match the special requirements of UWNs. The models are built upon machine learning techniques and trained with actual user data gathered from subjective tests. Our performance analysis shows how both of them can successfully estimate quality as a mean opinion score (MOS) value and, for the second model, even compute a distribution function for user scores.

Marginalization and health service coverage among indigenous, rural, and urban populations: a public health problem in Mexico.

INTRODUCTION:   Marginalization is a significant issue in Mexico, involving a lack of access to health services with differential impacts on Indigenous, rural and urban populations. The objective of this study was to understand Mexico’s public health problem across three population areas, Indigenous, rural and urban, in relation to degree of marginalization and health service coverage. METHODS:   The sampling universe of the study consisted of 107 458 geographic locations in the country. The study was retrospective, comparative and confirmatory. The study applied analysis of variance, parametric and non-parametric, correlation and correspondence analyses. RESULTS:   Significant differences were identified between the Indigenous, rural and urban populations with respect to their level of marginalization and access to health services. The most affected area was Indigenous, followed by rural areas. The sector that was least affected was urban. CONCLUSIONS:   Although health coverage is highly concentrated in urban areas in Mexico, shortages are mostly concentrated in rural areas where Indigenous groups represent the extreme end of marginalization and access to medical coverage. Inadequate access to health services in the Indigenous and rural populations throws the gravity of the public health problem into relief.

Quantum and Classical Molecular Dynamics of Ionic Liquid Electrolytes for Na/Li-based Batteries: Molecular Origins of the Conductivity Behavior.

Compositional effects on the charge-transport properties of electrolytes for batteries based on room-temperature ionic liquids (RTILs) are well-known. However, further understanding is required about the molecular origins of these effects, in particular regarding the replacement of Li by Na. In this work, we investigate the use of RTILs in batteries, by means of both classical molecular dynamics (MD), which provides information about structure and molecular transport, and ab initio molecular dynamics (AIMD), which provides information about structure. The focus has been placed on the effect of adding either Na(+) or Li(+) to 1-methyl-1-butyl-pyrrolidinium [C4 PYR](+) bis(trifluoromethanesulfonyl)imide [Tf2 N](-) . Radial distribution functions show excellent agreement between MD and AIMD, which ensures the validity of the force fields used in the MD. This is corroborated by the MD results for the density, the diffusion coefficients, and the total conductivity of the electrolytes, which reproduce remarkably well the experimental observations for all studied Na/Li concentrations. By extracting partial conductivities, it is demonstrated that the main contribution to the conductivity is that of [C4 PYR](+) and [Tf2 N](-) . However, addition of Na(+) /Li(+) , although not significant on its own, produces a dramatic decrease in the partial conductivities of the RTIL ions. The origin of this indirect effect can be traced to the modification of the microscopic structure of the liquid as observed from the radial distribution functions, owing to the formation of [Na(Tf2 N)n ]((n-1)-) and [Li(Tf2 N)n ]((n-1)-) clusters at high concentrations. This formation hinders the motion of the large ions, hence reducing the total conductivity. We demonstrate that this clustering effect is common to both Li and Na, showing that both ions behave in a similar manner at a microscopic level in spite of their distinct ionic radii. This is an interesting finding for extending Li-ion and Li-air technologies to their potentially cheaper Na-based counterparts.

DFT study of the effect of fluorine atoms on the crystal structure and semiconducting properties of poly(arylene-ethynylene) derivatives.

The effect of fluorine substitution on the molecular structure, crystal packing, and n-type semiconducting properties of a set of poly(arylene-ethynylene) polymers based on alternating thiadiazole and phenyl units linked through ethynylene groups has been studied by means of Density Functional Theory. As a result, an enlargement in the interplanar distance between cofacial polymer chains, as well as a decrease of the electronic coupling and electron mobility is predicted. On the other hand, fluorination could facilitate electron injection into the material. A polymer containing both alkoxy pendant chains and fluorine atoms is proposed as a compromise solution between efficiency of electron injection and charge transport within the material.