Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non­GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy.

BACKGROUND: Moyamoya angiopathy (MMA) is a rare cerebrovascular condition leading to stroke. Mutations in 15 genes have been identified in Mendelian forms of MMA, but they explain only a very small proportion of cases. Our aim was to investigate the genetic basis of MMA in consanguineous patients having unaffected parents in order to identify genes involved in autosomal recessive MMA. METHODS: Exome sequencing (ES) was performed in 6 consecutive consanguineous probands having MMA of unknown etiology. Functional consequences of variants were assessed using western blot and protein 3D structure analyses. RESULTS: Causative homozygous variants of NOS3, the gene encoding the endothelial nitric oxide synthase (eNOS), and GUCY1A3, the gene encoding the alpha1 subunit of the soluble guanylate cyclase (sGC) which is the major nitric oxide (NO) receptor in the vascular wall, were identified in 3 of the 6 probands. One NOS3 variant (c.1502 + 1G > C) involves a splice donor site causing a premature termination codon and leads to a total lack of eNOS in endothelial progenitor cells of the affected proband. The other NOS3 variant (c.1942 T > C) is a missense variant located into the flavodoxine reductase domain; it is predicted to be destabilizing and shown to be associated with a reduction of eNOS expression. The GUCY1A3 missense variant (c.1778G > A), located in the catalytic domain of the sGC, is predicted to disrupt the tridimensional structure of this domain and to lead to a loss of function of the enzyme. Both NOS3 mutated probands suffered from an infant-onset and severe MMA associated with posterior cerebral artery steno-occlusive lesions. The GUCY1A3 mutated proband presented an adult-onset MMA associated with an early-onset arterial hypertension and a stenosis of the superior mesenteric artery. None of the 3 probands had achalasia. CONCLUSIONS: We show for the first time that biallelic loss of function variants in NOS3 is responsible for MMA and that mutations in NOS3 and GUCY1A3 are causing fifty per cent of MMA in consanguineous patients. These data pinpoint the essential role of the NO pathway in MMA pathophysiology.

Virtual screening web servers: designing chemical probes and drug candidates in the cyberspace.

The interplay between life sciences and advancing technology drives a continuous cycle of chemical data growth; these data are most often stored in open or partially open databases. In parallel, many different types of algorithms are being developed to manipulate these chemical objects and associated bioactivity data. Virtual screening methods are among the most popular computational approaches in pharmaceutical research. Today, user-friendly web-based tools are available to help scientists perform virtual screening experiments. This article provides an overview of internet resources enabling and supporting chemical biology and early drug discovery with a main emphasis on web servers dedicated to virtual ligand screening and small-molecule docking. This survey first introduces some key concepts and then presents recent and easily accessible virtual screening and related target-fishing tools as well as briefly discusses case studies enabled by some of these web services. Notwithstanding further improvements, already available web-based tools not only contribute to the design of bioactive molecules and assist drug repositioning but also help to generate new ideas and explore different hypotheses in a timely fashion while contributing to teaching in the field of drug development.

Wood-density has no effect on stomatal control of leaf-level water use efficiency in an Amazonian forest.

Forest disturbances increase the proportion of fast-growing tree species compared to slow-growing ones. To understand their relative capacity for carbon uptake and their vulnerability to climate change, and to represent those differences in Earth system models, it is necessary to characterise the physiological differences in their leaf-level control of water use efficiency and carbon assimilation. We used wood density as a proxy for the fast-slow growth spectrum and tested the assumption that trees with a low wood density (LWD) have a lower water-use efficiency than trees with a high wood density (HWD). We selected 5 LWD tree species and 5 HWD tree species growing in the same location in an Amazonian tropical forest and measured in situ steady-state gas exchange on top-of-canopy leaves with parallel sampling and measurement of leaf mass area and leaf nitrogen content. We found that LWD species invested more nitrogen in photosynthetic capacity than HWD species, had higher photosynthetic rates and higher stomatal conductance. However, contrary to expectations, we showed that the stomatal control of the balance between transpiration and carbon assimilation was similar in LWD and HWD species and that they had the same dark respiration rates.

Machine learning-driven identification of drugs inhibiting cytochrome P450 2C9.

Cytochrome P450 2C9 (CYP2C9) is a major drug-metabolizing enzyme that represents 20% of the hepatic CYPs and is responsible for the metabolism of 15% of drugs. A general concern in drug discovery is to avoid the inhibition of CYP leading to toxic drug accumulation and adverse drug-drug interactions. However, the prediction of CYP inhibition remains challenging due to its complexity. We developed an original machine learning approach for the prediction of drug-like molecules inhibiting CYP2C9. We created new predictive models by integrating CYP2C9 protein structure and dynamics knowledge, an original selection of physicochemical properties of CYP2C9 inhibitors, and machine learning modeling. We tested the machine learning models on publicly available data and demonstrated that our models successfully predicted CYP2C9 inhibitors with an accuracy, sensitivity and specificity of approximately 80%. We experimentally validated the developed approach and provided the first identification of the drugs vatalanib, piriqualone, ticagrelor and cloperidone as strong inhibitors of CYP2C9 with IC values <18 µM and sertindole, asapiprant, duvelisib and dasatinib as moderate inhibitors with IC50 values between 40 and 85 µM. Vatalanib was identified as the strongest inhibitor with an IC50 value of 0.067 µM. Metabolism assays allowed the characterization of specific metabolites of abemaciclib, cloperidone, vatalanib and tarafenacin produced by CYP2C9. The obtained results demonstrate that such a strategy could improve the prediction of drug-drug interactions in clinical practice and could be utilized to prioritize drug candidates in drug discovery pipelines.

Dominant negative mutation in oxalate transporter SLC26A6 associated with enteric hyperoxaluria and nephrolithiasis.

BACKGROUND: Nephrolithiasis (NL) is a complex multifactorial disease affecting up to 10%-20% of the human population and causing a significant burden on public health systems worldwide. It results from a combination of environmental and genetic factors. Hyperoxaluria is a major risk factor for NL. METHODS: We used a whole exome-based approach in a patient with calcium oxalate NL. The effects of the mutation were characterised using cell culture and in silico analyses. RESULTS: We identified a rare heterozygous missense mutation (c.1519C>T/p.R507W) in the SLC26A6 gene that encodes a secretory oxalate transporter. This mutation cosegregated with hyperoxaluria in the family. In vitro characterisation of mutant SLC26A6 demonstrated that Cl--dependent oxalate transport was dramatically reduced because the mutation affects both SLC26A6 transport activity and membrane surface expression. Cotransfection studies demonstrated strong dominant-negative effects of the mutant on the wild-type protein indicating that the phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone. CONCLUSION: Our study is in line with previous observations made in the mouse showing that SLC26A6 inactivation can cause inherited enteric hyperoxaluria with calcium oxalate NL. Consistent with an enteric form of hyperoxaluria, we observed a beneficial effect of increasing calcium in the patient's diet to reduce urinary oxalate excretion.

Antibacterial activity of barbatimão (Stryphnodendron adstringens) against periodontopathogens and cytotoxic effects on fibroblasts.

This in vitro study aimed to evaluate the growth-inhibitory effects against periodontal disease-causing bacteria and cytotoxic effects against mouse fibroblast cells of the Stryphnodendron adstringens (barbatimão) hydroalcoholic extract. The contents of phenols and tannins in the extract were determined. The growth-inhibitory activity of the barbatimão was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The viability of fibroblast cells was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay at 24 and 48 h post-treatment. The MIC values of the extract against Prevotella intermedia, Porphyromonas gingivalis, and Fusobacterium nucleatum were 0.05, 0.125, and 2 mg mL-1, respectively, while the MBC values were 4, 2, and 2 mg mL-1, respectively. The viability rate of barbatimão (0.25 mg mL-1)-treated L929 cells was higher than that of chlorhexidine (0.12%)-treated L929 cells at 48 h post-treatment. The contents of total phenolics and total tannins in the extract were 837.39 ± 0.10 and 785.82 ± 0.14 mg of tannic acid equivalent per gram of the extract, respectively. These findings indicate that the barbatimão hydroalcoholic extract, which exerted potent growth-inhibitory effects against the test microbial species and low cytotoxic effects on fibroblasts, has potential applications in the development of novel mouthwash products.

A dietary spray-dried plasma feeding programme improves growth performance and reduces faecal bacterial shedding of nursery pigs challenged with enterotoxigenic Escherichia coli K88.

This study aimed to assess the effects of different spray-dried plasma (SDP) feeding programmes to pigs on performance, intestinal histomorphology and faecal bacterial shedding after an Escherichia coli K88 challenge. A total of 96 piglets (5.77 ± 0.01 kg) were weaned at 21 days of age (Day 0) and challenged with 3 ml of 1 × 1010 CFU of E. coli K88 in total 3.0 × 1010 CFU/animal on Days 0, 2 and 4. Pigs were fed nursery diets containing 0.0%, 3.0%, 6.0% or 9.0% SDP from weaning to 35 days of age; 0.0%, 1.5%, 3.0% or 4.5% SDP from 36 to 49 days; and the same control diet (without SDP), for the last 10 days of the experiment (50-59 days of age). Performance was measured from 35 to 59 days of age and faecal bacterial shedding and intestinal histomorphometry were evaluated at Days 28 and 49 of age respectively. From 21 to 35 days of age, there was a linear effect for body weight (BW) and average daily gain (ADG), a trend of linear effect for average daily feed intake (ADFI) and a quadratic effect for feed:gain ratio (FG). From 21 to 49 days, the 9.0:4.5% and 6.0:3.0% SDP feeding programmes improved BW, ADG and FG when compared to the other treatments. At 59 days of age, BW and ADG were increased by the two highest SDP feeding programmes. The 9.0:4.5% SDP feeding programme increased ADFI from 21 to 59 days of age, with 6.0:3.0% being intermediate and the other two treatments being lowest. The CFU counts of E. coli/g of faeces decreased linearly with increasing addition of SDP. These results indicate that an extended inclusion of increased SDP levels in post-weaning diets can improve growth potential and decrease bacterial shedding induced by E. coli K88.

Using Satellite Observations to Evaluate Model Microphysical Representation of Arctic Mixed-Phase Clouds.

Mixed-phase clouds play an important role in determining Arctic warming, but are parametrized in models and difficult to constrain with observations. We use two satellite-derived cloud phase metrics to investigate the vertical structure of Arctic clouds in two global climate models that use the Community Atmosphere Model version 6 (CAM6) atmospheric component. We report a model error limiting ice nucleation, produce a set of Arctic-constrained model runs by adjusting model microphysical variables to match the cloud phase metrics, and evaluate cloud feedbacks for all simulations. Models in this small ensemble uniformly overestimate total cloud fraction in the summer, but have variable representation of cloud fraction and phase in the winter and spring. By relating modeled cloud phase metrics and changes in low-level liquid cloud amount under warming to longwave cloud feedback, we show that mixed-phase processes mediate the Arctic climate by modifying how wintertime and springtime clouds respond to warming.

Light induces peridinin and docosahexaenoic acid accumulation in the dinoflagellate Durusdinium glynnii.

Peridinin is a light-harvesting carotenoid present in phototrophic dinoflagellates and has great potential for new drug applications and cosmetics development. Herein, the effects of irradiance mediated by light-emitting diodes on growth performance, carotenoid and fatty acid profiles, and antioxidant activity of the endosymbiotic dinoflagellate Durusdinium glynnii were investigated. The results demonstrate that D. glynnii is particularly well adapted to low-light conditions; however, it can be high-light-tolerant. In contrast to other light-harvesting carotenoids, the peridinin accumulation in D. glynnii occurred during high-light exposure. The peridinin to chlorophyll-a ratio varied as a function of irradiance, while the peridinin to total carotenoids ratio remained stable. Under optimal irradiance for growth, there was a peak in docosahexaenoic acid (DHA) bioaccumulation. This study contributes to the understanding of the photoprotective role of peridinin in endosymbiont dinoflagellates and highlights the antioxidant activity of peridinin-rich extracts. KEY POINTS: • Peridinin has a protective role against chlorophyll photo-oxidation • High light conditions induce cellular peridinin accumulation • D. glynnii accumulates high amounts of DHA under optimal light supply.

A Hybrid Docking and Machine Learning Approach to Enhance the Performance of Virtual Screening Carried out on Protein-Protein Interfaces.

The modulation of protein-protein interactions (PPIs) by small chemical compounds is challenging. PPIs play a critical role in most cellular processes and are involved in numerous disease pathways. As such, novel strategies that assist the design of PPI inhibitors are of major importance. We previously reported that the knowledge-based DLIGAND2 scoring tool was the best-rescoring function for improving receptor-based virtual screening (VS) performed with the Surflex docking engine applied to several PPI targets with experimentally known active and inactive compounds. Here, we extend our investigation by assessing the vs. potential of other types of scoring functions with an emphasis on docking-pose derived solvent accessible surface area (SASA) descriptors, with or without the use of machine learning (ML) classifiers. First, we explored rescoring strategies of Surflex-generated docking poses with five GOLD scoring functions (GoldScore, ChemScore, ASP, ChemPLP, ChemScore with Receptor Depth Scaling) and with consensus scoring. The top-ranked poses were post-processed to derive a set of protein and ligand SASA descriptors in the bound and unbound states, which were combined to derive descriptors of the docked protein-ligand complexes. Further, eight ML models (tree, bagged forest, random forest, Bayesian, support vector machine, logistic regression, neural network, and neural network with bagging) were trained using the derivatized SASA descriptors and validated on test sets. The results show that many SASA descriptors are better than Surflex and GOLD scoring functions in terms of overall performance and early recovery success on the used dataset. The ML models were superior to all scoring functions and rescoring approaches for most targets yielding up to a seven-fold increase in enrichment factors at 1% of the screened collections. In particular, the neural networks and random forest-based ML emerged as the best techniques for this PPI dataset, making them robust and attractive vs. tools for hit-finding efforts. The presented results suggest that exploring further docking-pose derived SASA descriptors could be valuable for structure-based virtual screening projects, and in the present case, to assist the rational design of small-molecule PPI inhibitors.

Enuresis and upper airway obstruction: BNP and ADH hormones behavior before and after airway surgery.

INTRODUCTION: Upper airway obstruction (UAO) is a common condition in all pediatric population, with a 27% prevalence. Primary monosymptomatic nocturnal enuresis (PMNE) is a condition related to UAO in 8% to 47% of these children. The specific pathophysiological mechanism of this bond is not well understood. Some authors suggest a connection between brain natrituretic peptide (BNP) and anti-diuretic hormone (ADH) during sleep. The aim of this study was to evaluate hormone profile (ADH and BNP) and improvement in dry nights in a sample of children before and after surgical treatment of the UAO. METHODS: This is a longitudinal prospective interventionist study in children, 5 to 14 years of age, with UAO and PMNE recruited in a specialty outpatient clinic. Children presenting UAO and PMNE were evaluated with a 30-day dry night diary and blood samples were collected to evaluate ADH and BNP before and after upper airway surgery. Data were analyzed prior to surgery and 90-120 days after surgery. RESULTS: Twenty-one children with a mean age of 9.7 years were included. Mean BNP before surgery was 116.5 ± 126.5 pg/mL and 156.2 ± 112.3 pg/mL after surgery (p<0.01). Mean ADH was 5.8 ± 3.2 pg/mL and 14.6 ± 35.4 before and after surgery, respectively (p=0.26). The percentage of dry nights went from 32.3 ± 24.7 before surgery to 75.4 ± 33.4 after surgery (p˂0.01). CONCLUSION: Surgery for airway obstruction contributed to an increase in BNP without increasing ADH. A total of 85.8% of the children presented partial or complete improvement of their enuresis.

FastTargetPred: a program enabling the fast prediction of putative protein targets for input chemical databases.

SUMMARY: Several web-based tools predict the putative targets of a small molecule query compound by similarity to molecules with known bioactivity data using molecular fingerprints. In numerous situations, it would however be valuable to be able to run such computations on a local computer. We present FastTargetPred, a new program for the prediction of protein targets for small molecule queries. Structural similarity computations rely on a large collection of confirmed protein-ligand activities extracted from the curated ChEMBL 25 database. The program allows to annotate an input chemical library of ∼100k compounds within a few hours on a simple personal computer. AVAILABILITY AND IMPLEMENTATION: FastTargetPred is written in Python 3 (≥3.7) and C languages. Python code depends only on the Python Standard Library. The program can be run on Linux, MacOS and Windows operating systems. Pre-compiled versions are available at https://github.com/ludovicchaput/FastTargetPred. FastTargetPred is licensed under the GNU GPLv3. The program calls some scripts from the free chemistry toolkit MayaChemTools. CONTACT: bruno.villoutreix@inserm.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Anti-Factor B Antibodies and Acute Postinfectious GN in Children.

BACKGROUND: The pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that, unlike acute postinfectious GN, has a poor prognosis. METHODS: This retrospective study investigated mechanisms of complement activation in 34 children with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement protein autoantibodies, carried out related functional characterization, and compared results with those of 60 children from the National French Registry who had C3 glomerulopathy and persistent hypocomplementemia. RESULTS: All children with acute postinfectious GN had activation of the alternative pathway of the complement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3 convertase) were found in a significantly higher proportion of children with the disorder versus children with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase activity in vitro, confirming their pathogenic effect. We also identified crucial antibody binding sites on factor B, including one correlated to disease severity. CONCLUSIONS: These findings elucidate the pathophysiologic mechanisms underlying acute postinfectious GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as C3 glomerulopathy, and to help determine therapy.

Primary conjunctival sporotrichosis in three cats from Northeastern Brazil.

INTRODUCTION: Classically, sporotrichosis occurs as a chronic granulomatous lymphocutaneous infection. The extracutaneous form is uncommon and may affect the eye without cutaneous involvement. The most frequent form of ocular sporotrichosis reported in humans is a granulomatous conjunctivitis. There are no previous reports on primary ocular sporotrichosis in cats. PROCEDURES: Three mixed breed cats rescued from shelters were referred by the veterinarian for ophthalmic evaluation with a complaint of conjunctivitis nonresponsive to treatment with no evidence of skin disease or systemic disease. Complete ophthalmic examination, conjunctival cytology, and microbiological analysis were performed. RESULTS: Ophthalmic examinations revealed epiphora, purulent ocular discharge, conjunctival hyperemia, and a mass in the palpebral conjunctiva. Conjunctival cytology revealed segmented and degenerated neutrophils, conjunctival epithelial cells, and an abundant number of round and oval cells compatible with Sporothrix spp. Microbiological culture was performed and confirmed the presence of fungi from the Sporothrix schenckii complex. All animals were treated with oral itraconazole; two animals received topical itraconazole in association with oral treatment. Case 1 was refractory to treatment, and iodate potassium was combined with itraconazole therapy without resolution at the time of this publication. Cases 2 and 3 had complete resolution of conjunctival lesions with four months of oral and topical itraconazole therapy. CONCLUSION: Conjunctival sporotrichosis should be considered as a differential diagnosis of conjunctivitis in cats from endemic regions. Conjunctival cytology is an important tool that can aid early diagnosis.

In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2-Spike RBD Interface.

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike-ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure-function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.

Computational Analysis of Chemical Space of Natural Compounds Interacting with Sulfotransferases.

The aim of this study was to investigate the chemical space and interactions of natural compounds with sulfotransferases (SULTs) using ligand- and structure-based in silico methods. An in-house library of natural ligands (hormones, neurotransmitters, plant-derived compounds and their metabolites) reported to interact with SULTs was created. Their chemical structures and properties were compared to those of compounds of non-natural (synthetic) origin, known to interact with SULTs. The natural ligands interacting with SULTs were further compared to other natural products for which interactions with SULTs were not known. Various descriptors of the molecular structures were calculated and analyzed. Statistical methods (ANOVA, PCA, and clustering) were used to explore the chemical space of the studied compounds. Similarity search between the compounds in the different groups was performed with the ROCS software. The interactions with SULTs were additionally analyzed by docking into different experimental and modeled conformations of SULT1A1. Natural products with potentially strong interactions with SULTs were outlined. Our results contribute to a better understanding of chemical space and interactions of natural compounds with SULT enzymes and help to outline new potential ligands of these enzymes.

Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis.

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.

Fast Rescoring Protocols to Improve the Performance of Structure-Based Virtual Screening Performed on Protein-Protein Interfaces.

Protein-protein interactions (PPIs) are attractive targets for drug design because of their essential role in numerous cellular processes and disease pathways. However, in general, PPIs display exposed binding pockets at the interface, and as such, have been largely unexploited for therapeutic interventions with low-molecular weight compounds. Here, we used docking and various rescoring strategies in an attempt to recover PPI inhibitors from a set of active and inactive molecules for 11 targets collected in ChEMBL and PubChem. Our focus is on the screening power of the various developed protocols and on using fast approaches so as to be able to apply such a strategy to the screening of ultralarge libraries in the future. First, we docked compounds into each target using the fast "pscreen" mode of the structure-based virtual screening (VS) package Surflex. Subsequently, the docking poses were postprocessed to derive a set of 3D topological descriptors: (i) shape similarity and (ii) interaction fingerprint similarity with a co-crystallized inhibitor, (iii) solvent-accessible surface area, and (iv) extent of deviation from the geometric center of a reference inhibitor. The derivatized descriptors, together with descriptor-scaled scoring functions, were utilized to investigate possible impacts on VS performance metrics. Moreover, four standalone scoring functions, RF-Score-VS (machine-learning), DLIGAND2 (knowledge-based), Vinardo (empirical), and X-SCORE (empirical), were employed to rescore the PPI compounds. Collectively, the results indicate that the topological scoring algorithms could be valuable both at a global level, with up to 79% increase in areas under the receiver operating characteristic curve for some targets, and in early stages, with up to a 4-fold increase in enrichment factors at 1% of the screened collections. Outstandingly, DLIGAND2 emerged as the best scoring function on this data set, outperforming all rescoring techniques in terms of VS metrics. The described methodology could help in the rational design of small-molecule PPI inhibitors and has direct applications in many therapeutic areas, including cancer, CNS, and infectious diseases such as COVID-19.

Diagnostic Validity of an Isokinetic Testing to Identify Partial Anterior Cruciate Ligament Injuries.

OBJECTIVE: To assess the diagnostic validity of an isokinetic testing to detect partial injuries on the anterior cruciate ligament (ACL). DESIGN: Prospective diagnostic study. SETTINGS: Orthopedic clinic, physiotherapy clinic, orthopedic hospital, and diagnostic/image clinic. PARTICIPANTS: Consecutive patients (n = 29) with unilateral knee complaint submitted to physical examination, magnetic resonance images (MRIs), and isokinetic testing prior to surgery of ACL reconstruction. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The isokinetic torque curves data from extensor and flexor muscles were converted to frequency domain by fast Fourier transformation and compared with healthy contralateral limb. Differences were categorized as unstable knees and these conclusions were compared with patient's physical examinations (doctor's conclusion on ACL integrity) and MRIs (as the radiologist conclusions on ACL integrity). After surgery, all intraoperatively confirmed partial injured patient's data were collected. The diagnostic accuracy measures to compare the conclusions of all 3 professionals included sensitivity, specificity, positive predictive value, negative predictive value, disease prevalence, positive likelihood ratio, and accuracy-all using a confidence interval of 95%. RESULTS: Compared with MRI, the sensitivity of isokinetic test for an ACL partial injury was 90.00%, specificity 83.33%, positive predictive value 52.94%, negative predictive value 97.56%, and accuracy 84.48%. Compared with physical examination, the sensitivity of isokinetic test for an ACL partial injury was 85.71%, specificity 78.43%, positive predictive value 35.29%, negative predictive value 97.56%, and accuracy 79.31%. CONCLUSIONS: This method of isokinetic data analysis through fast Fourier transformation can be used to improve diagnostic accuracy of a difficult detection injury. Even present, a partial ACL injury can produce a stable knee during isokinetic testing and could be used to detect candidates for conservative treatment based on strengthening exercises, reducing surgery risks, and financial and social impact on patient's life.