Harnessing conformational dynamics in enzyme catalysis to achieve nature-like catalytic efficiencies: the shortest path map tool for computational enzyme redesign.

Enzymes exhibit diverse conformations, as represented in the free energy landscape (FEL). Such conformational diversity provides enzymes with the ability to evolve towards novel functions. The challenge lies in identifying mutations that enhance specific conformational changes, especially if located in distal sites from the active site cavity. The shortest path map (SPM) method, which we developed to address this challenge, constructs a graph based on the distances and correlated motions of residues observed in nanosecond timescale molecular dynamics (MD) simulations. We recently introduced a template based AlphaFold2 (tAF2) approach coupled with 10 nanosecond MD simulations to quickly estimate the conformational landscape of enzymes and assess how the FEL is shifted after mutation. In this study, we evaluate the potential of SPM when coupled with tAF2-MD in estimating conformational heterogeneity and identifying key conformationally-relevant positions. The selected model system is the beta subunit of tryptophan synthase (TrpB). We compare how the SPM pathways differ when integrating tAF2 with different MD simulation lengths from as short as 10 ns until 50 ns and considering two distinct Amber forcefield and water models (ff14SB/TIP3P versus ff19SB/OPC). The new methodology can more effectively capture the distal mutations found in laboratory evolution, thus showcasing the efficacy of tAF2-MD-SPM in rapidly estimating enzyme dynamics and identifying the key conformationally relevant hotspots for computational enzyme engineering.

Health-related quality of life in nonvalvular atrial fibrillation patients with controlled or uncontrolled anticoagulation status.

BACKGROUND: There is a dearth of evidence regarding Health-Related Quality of Life (HRQoL) in nonvalvular atrial fibrillation (NVAF) patients undergoing oral anticoagulation therapy. Our objective was to describe HRQoL in NVAF patients on oral anticoagulation, focusing on uncontrolled patients on vitamin K antagonists (VKAs) versus controlled patients on VKAs or non-vitamin K antagonist oral anticoagulants (NOACs), in a real-world setting. Additionally, we assessed the clinical characteristics of patients with uncontrolled anticoagulation. METHODS: An observational, multicentre, and cross-sectional study, enrolling 38 Spanish Hospitals' Internal Medicine Departments. HRQoL was assessed using the validated Spanish version of the Sawicki questionnaire. High self-perceived HRQoL was indicated by high scores in the general treatment satisfaction and self-efficacy dimensions, and by low scores in the strained social network, daily hassles and distress dimensions. RESULTS: Five hundred and one patients were included for assessment. Mean scores ± SD were closer to a high perceived HRQoL in controlled than uncontrolled patients for the five dimensions of the questionnaire: 4.9 ± 1.0 versus 3.6 ± 1.3 for general treatment satisfaction; 4.3 ± 1.0 versus 3.6 ± 1.0 for self-efficacy, 3.1 ± 0.9 versus 3.9 ± 1.1 for strained social network, 2.1 ± 0.8 versus 3.0 ± 1.0 for daily hassles and 1.8 ± 0.9 versus 2.6 ± 1.2 for distress. CONCLUSIONS: HRQoL in patients with controlled anticoagulant status treated with NOACs or VKAs was better than in patients with uncontrolled anticoagulant status. This seems to indicate that anticoagulation control status influences perception of HRQoL, highlighting the importance of its evaluation when assessing HRQoL in NVAF patients.

The shortest path method (SPM) webserver for computational enzyme design.

SPMweb is the online webserver of the Shortest Path Map (SPM) tool for identifying the key conformationally-relevant positions of a given enzyme structure and dynamics. The server is built on top of the DynaComm.py code and enables the calculation and visualization of the SPM pathways. SPMweb is easy-to-use as it only requires three input files: the three-dimensional structure of the protein of interest, and the two matrices (distance and correlation) previously computed from a Molecular Dynamics simulation. We provide in this publication information on how to generate the files for SPM construction even for non-expert users and discuss the most relevant parameters that can be modified. The tool is extremely fast (it takes less than one minute per job), thus allowing the rapid identification of distal positions connected to the active site pocket of the enzyme. SPM applications expand from computational enzyme design, especially if combined with other tools to identify the preferred substitution at the identified position, but also to rationalizing allosteric regulation, and even cryptic pocket identification for drug discovery. The simple user interface and setup make the SPM tool accessible to the whole scientific community. SPMweb is freely available for academia at http://spmosuna.com/.

Remote Monitoring and Data Collection for Decentralized Clinical Trials.

Importance: Less than 5% of patients with cancer enroll in a clinical trial, partly due to financial and logistic burdens, especially among underserved populations. The COVID-19 pandemic marked a substantial shift in the adoption of decentralized trial operations by pharmaceutical companies. Objective: To assess the current global state of adoption of decentralized trial technologies, understand factors that may be driving or preventing adoption, and highlight aspirations and direction for industry to enable more patient-centric trials. Design, Setting, and Participants: The Bloomberg New Economy International Cancer Coalition, composed of patient advocacy, industry, government regulator, and academic medical center representatives, developed a survey directed to global biopharmaceutical companies of the coalition from October 1 through December 31, 2022, with a focus on registrational clinical trials. The data for this survey study were analyzed between January 1 and 31, 2023. Exposure: Adoption of decentralized clinical trial technologies. Main Outcomes and Measures: The survey measured (1) outcomes of different remote monitoring and data collection technologies on patient centricity, (2) adoption of these technologies in oncology and all therapeutic areas, and (3) barriers and facilitators to adoption using descriptive statistics. Results: All 8 invited coalition companies completed the survey, representing 33% of the oncology market by revenues in 2021. Across nearly all technologies, adoption in oncology trials lags that of all trials. In the current state, electronic diaries and electronic clinical outcome assessments are the most used technology, with a mean (SD) of 56% (19%) and 51% (29%) adoption for all trials and oncology trials, respectively, whereas visits within local physician networks is the least adopted at a mean (SD) of 12% (18%) and 7% (9%), respectively. Looking forward, the difference between the current and aspired adoption rate in 5 years for oncology is large, with respondents expecting a 40% or greater absolute adoption increase in 8 of the 11 technologies surveyed. Furthermore, digitally enabled recruitment, local imaging capabilities, and local physician networks were identified as technologies that could be most effective for improving patient centricity in the long term. Conclusions and Relevance: These findings may help to galvanize momentum toward greater adoption of enabling technologies to support a new paradigm of trials that are more accessible, less burdensome, and more inclusive.

AlphaFold2 and Deep Learning for Elucidating Enzyme Conformational Flexibility and Its Application for Design.

The recent success of AlphaFold2 (AF2) and other deep learning (DL) tools in accurately predicting the folded three-dimensional (3D) structure of proteins and enzymes has revolutionized the structural biology and protein design fields. The 3D structure indeed reveals key information on the arrangement of the catalytic machinery of enzymes and which structural elements gate the active site pocket. However, comprehending enzymatic activity requires a detailed knowledge of the chemical steps involved along the catalytic cycle and the exploration of the multiple thermally accessible conformations that enzymes adopt when in solution. In this Perspective, some of the recent studies showing the potential of AF2 in elucidating the conformational landscape of enzymes are provided. Selected examples of the key developments of AF2-based and DL methods for protein design are discussed, as well as a few enzyme design cases. These studies show the potential of AF2 and DL for allowing the routine computational design of efficient enzymes.

Estimating conformational heterogeneity of tryptophan synthase with a template-based Alphafold2 approach.

The three-dimensional structure of the enzymes provides very relevant information on the arrangement of the catalytic machinery and structural elements gating the active site pocket. The recent success of the neural network Alphafold2 in predicting the folded structure of proteins from the primary sequence with high levels of accuracy has revolutionized the protein design field. However, the application of Alphafold2 for understanding and engineering function directly from the obtained single static picture is not straightforward. Indeed, understanding enzymatic function requires the exploration of the ensemble of thermally accessible conformations that enzymes adopt in solution. In the present study, we evaluate the potential of Alphafold2 in assessing the effect of the mutations on the conformational landscape of the beta subunit of tryptophan synthase (TrpB). Specifically, we develop a template-based Alphafold2 approach for estimating the conformational heterogeneity of several TrpB enzymes, which is needed for enhanced stand-alone activity. Our results show the potential of Alphafold2, especially if combined with molecular dynamics simulations, for elucidating the changes induced by mutation in the conformational landscapes at a rather reduced computational cost, thus revealing its plausible application in computational enzyme design.

Pancreatic endocrine tumors or apudomas.

INTRODUCTION AND OBJECTIVE: pancreatic endocrine tumors (PET) are difficult to diagnose. Their accurate localization using imaging techniques is intended to provide a definite cure. The goal of this retrospective study was to review a PET series from a private institution. PATIENTS AND METHODS: the medical records of 19 patients with PETs were reviewed, including 4 cases of MEN-1, for a period of 17 years (1994-2010). A database was set up with ten parameters: age, sex, symptoms, imaging techniques, size and location in the pancreas, metastasis, surgery, complications, adjuvant therapies, definite diagnosis, and survival or death. RESULTS: a total of 19 cases were analyzed. Mean age at presentation was 51 years (range: 26-67 y) (14 males, 5 females), and tumor size was 5 to 80 mm (X: 20 mm). Metastatic disease was present in 37% (7/19). Most underwent the following imaging techniques: ultrasounds, computed tomography (CT) an magnetic resonance imaging (MRI). Fine needle aspiration punction (FNA) was performed for the primary tumor in 4 cases. Non-functioning: 7 cases (37%), insulinoma: 2 cases [1 with possible multiple endocrine neoplasia (MEN)], Zollinger-Ellison syndrome (ZES) from gastrinoma: 5 (3 with MEN-1), glucagonoma: 2 cases, 2 somatostatinomas; carcinoid: 1 case with carcinoide-like syndrome. Most patients were operated upon: 14/19 (73%). Four (4/14:28%) has postoperative complications following pancreatectomy: pancreatitis, pseudocyst, and abdominal collections. Some patients received chemotherapy (4), somatostatin (3) and interferon (2) before or after surgery. Median follow-up was 48 months. Actuarial survival during the study was 73.6% (14/19). CONCLUSIONS: age was similar to that described in the literature. Males were predominant. Most cases were non-functioning (37%). Most patients underwent surgery (73%) with little morbidity (28%) and an actuarial survival of 73.6% at the time of the study.

Correlation Between Early Endpoints and Overall Survival in Non-Small-Cell Lung Cancer: A Trial-Level Meta-Analysis.

Early endpoints, such as progression-free survival (PFS), are increasingly used as surrogates for overall survival (OS) to accelerate approval of novel oncology agents. Compiling trial-level data from randomized controlled trials (RCTs) could help to develop a predictive framework to ascertain correlation trends between treatment effects for early and late endpoints. Through trial-level correlation and random-effects meta-regression analysis, we assessed the relationship between hazard ratio (HR) OS and (1) HR PFS and (2) odds ratio (OR) PFS at 4 and 6 months, stratified according to the mechanism of action of the investigational product. Using multiple source databases, we compiled a data set including 81 phase II-IV RCTs (35 drugs and 156 observations) of patients with non-small-cell lung cancer. Low-to-moderate correlations were generally observed between treatment effects for early endpoints (based on PFS) and HR OS across trials of agents with different mechanisms of action. Moderate correlations were seen between treatment effects for HR PFS and HR OS across all trials, and in the programmed cell death-1/programmed cell death ligand-1 and epidermal growth factor receptor trial subsets. Although these results constitute an important step, caution is advised, as there are some limitations to our evaluation, and an additional patient-level analysis would be needed to establish true surrogacy.

Three-layered repair with a collagen membrane and a mucosal rotational flap reinforced with fibrine for palatal fistula closure in children.

The incidence of postoperative fistula following primary cleft palate repair ranges from 0% to 58%. The reported recurrence rate is between 33% and 37%, and the prognosis of a successful closure declines with each reoperation. Closure of palatal fistulas can be achieved by different techniques depending on its size and the experience of the surgeon. Local, regional and distant flaps are commonly used. Alternatively, or in addition to the previous ones, synthetic materials are becoming very popular nowadays. However, a scarcity of articles explains in detail a simple and effective method in children. We present a case report and the procedure proposed by our pediatric surgery team consisting of a three-layered repair, with a collagen membrane placed over the reconstructed nasal mucosa, and a rotational palatal mucosa flap reinforced with a fibrine sealant. This method is simple, easy to reproduce, effective and has a low rate of complications.

Interactive Effect of UVR and Phosphorus on the Coastal Phytoplankton Community of the Western Mediterranean Sea: Unravelling Eco-Physiological Mechanisms.

Some of the most important effects of global change on coastal marine systems include increasing nutrient inputs and higher levels of ultraviolet radiation (UVR, 280-400 nm), which could affect primary producers, a key trophic link to the functioning of marine food webs. However, interactive effects of both factors on the phytoplankton community have not been assessed for the Mediterranean Sea. An in situ factorial experiment, with two levels of ultraviolet solar radiation (UVR+PAR vs. PAR) and nutrients (control vs. P-enriched), was performed to evaluate single and UVR×P effects on metabolic, enzymatic, stoichiometric and structural phytoplanktonic variables. While most phytoplankton variables were not affected by UVR, dissolved phosphatase (APAEX) and algal P content increased in the presence of UVR, which was interpreted as an acclimation mechanism of algae to oligotrophic marine waters. Synergistic UVR×P interactive effects were positive on photosynthetic variables (i.e., maximal electron transport rate, ETRmax), but negative on primary production and phytoplankton biomass because the pulse of P unmasked the inhibitory effect of UVR. This unmasking effect might be related to greater photodamage caused by an excess of electron flux after a P pulse (higher ETRmax) without an efficient release of carbon as the mechanism to dissipate the reducing power of photosynthetic electron transport.

Imaging on pelvic pyomyositis in children related to pathogenesis.

PURPOSE: Pelvic pyomyositis in children is a rare infectious condition, although it is increasingly reported in temperate climates. Often considered a primary disease, new diagnostic methods are able to identify additional foci of infection. The purpose of this study is to review our patients and to analyze the imaging studies to determine its pathogenesis. METHODS: A retrospective study of the clinical charts and imaging records of 11 patients was made, noting the number and location of muscles involved, as well as bone and joint involvement. RESULTS: Besides the classical form of pelvic pyomyositis, i.e., iliopsoas pyomyositis, other muscular groups were frequently affected, often with multiple involvement. Bone involvement is also frequent. Magnetic resonance imaging (MRI) gives the most useful information. CONCLUSION: MRI is the diagnostic procedure of choice for diagnosing pelvic pyomyositis in children. It may also have an elucidating role in the debated pathogenesis of this condition. In most of the cases, pelvic pyomyositis in children could be secondary.

Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid.

BACKGROUND/AIMS: The sensitivity of fetal rat liver to maternal obstructive cholestasis during pregnancy (OCP), and the effect of ursodeoxycholic acid (UDCA) were investigated. METHODS: UDCA was administered (i.g. 0.6 mg/kg b.wt./day) from day 14 to day 21 of pregnancy after maternal common bile duct ligation. RESULTS: Impairment in the activity of antioxidant enzymes, levels of total glutathione and GSH/GSSG ratio and the degrees of lipid peroxidation and protein carbonylation were similar in livers of OCP mothers and fetuses at term, despite hypercholanemia was milder in fetuses. Treatment of OCP rats with UDCA reduced maternal and fetal liver oxidative stress. Although maternal hypercholanemia was not corrected, fetal serum concentrations of major bile acids (except UDCA and beta-muricholic acid) were reduced. Fetal liver expression of key enzyme in bile acid synthesis, Cyp7a1, Cyp27 and Cyp8b1 was not affected by OCP or UDCA treatment. In OCP fetal livers, the relative expression of Bax-alpha and Bcl-2 and the activity of caspase-3, but not caspase-8, were increased. These changes were markedly reduced in fetuses of OCP animals treated with UDCA. CONCLUSIONS: OCP induced moderate fetal hypercholanemia but marked liver oxidative stress and apoptosis that were partly prevented by treatment of pregnant rats with UDCA.

Autocrine IL-4 gene regulation at late phases of TCR activation in differentiated Th2 cells.

IL-4 is a multifunctional cytokine whose secretion displays important immunomodulatory functions. Its expression is regulated at the level of transcription, and one of the main factors involved is NFAT. The IL-4-induced transcription factor Stat6 is required for the development of naive T cells into Th2 phenotype, capable of secreting IL-4. However, IL-4 production by differentiated Th2 cells is IL-4 independent; thus, it remains unclear whether Stat6 plays any role in the IL-4 expression by mature Th2 cells. We have analyzed in the Th2 clone D10.G4.1 the nuclear proteins able to bind the regulatory element P1 of the IL-4 promoter. Gel-shift assays show NFAT1 as the most abundant nuclear protein that binds to P1 after ionomycin plus PMA activation, whereas Stat6 accounts for the bulk of the P1 binding in the presence of exogenous IL-4. Reporter experiments agree with an inhibitory effect of Stat6 on the NFAT1-induced transcriptional activity directed by the P1 element. CD3 signaling leads to an early induction of NFAT1-P1 complexes correlating with a strong induction of the IL-4 gene. In later phases of CD3 activation, P1 is also bound by Stat6 and a fall in the IL-4 mRNA levels takes place. These two late events during CD3 activation were found to be sensible in experiments conducted with an anti-IL-4 Ab. These results suggest that IL-4 endogenously produced by Th2 cells under TCR triggering modulates its own expression through Stat6.

Tumores oculares: incidencia en el Hospital Carlos Andrade Marín

Los tumores que afectan al globo ocular y sus anexos pueden ser benignos o malignos y por sus características de rpesentación, inducen una presunción al oftalmólogo. El diagnóstico y tratamiento oportunos evitan que la lesión continúe con su evolución natural o el desarrollo de complicaciones. Varios exámenes corroboran el diagnóstico clínico, entre los que se incluyen la ecografía, angiografía con fluorecceína, tomografía computarizada y resonancia magnética, sin embargo, la biopsia y el estudio histopatológico determinan con certeza el tipo de tumor.