Synthesis of LAVR-289, a new [(Z)-3-(acetoxymethyl)-4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug with pronounced antiviral activity against DNA viruses.

New acyclic pyrimidine nucleoside phosphonate prodrugs with a 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid skeleton (O-DAPy nucleobase) were prepared through a convergent synthesis by olefin cross-metathesis as the key step. Several acyclic nucleoside 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug exhibited in vitro antiviral activity in submicromolar or nanomolar range against varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus type 1 (HSV-1) and type 2 (HSV-2), and vaccinia virus (VV), with good selective index (SI). Among them, the analogue 9c (LAVR-289) proved markedly inhibitory against VZV wild-type (TK+) (EC50 0.0035 µM, SI 740) and for thymidine kinase VZV deficient strains (EC50 0.018 µM, SI 145), with a low morphological toxicity in cell culture at 100 µM and acceptable cytostatic activity resulting in excellent selectivity. Compound 9c exhibited antiviral activity against HCMV (EC50 0.021 µM) and VV (EC50 0.050 µM), as well as against HSV-1 (TK-) (EC50 0.0085 µM). Finally, LAVR-289 (9c) deserves further (pre)clinical investigations as a potent candidate broad-spectrum anti-herpesvirus drug.

Drivers of employment dynamics of AI innovators.

Based on an analysis of companies developing artificial intelligence (AI) technologies, this study offers fresh evidence on the role of innovation as one of the drivers of employment growth. GMM-SYS estimates on a worldwide longitudinal dataset covering 4,184 firms that patented inventions involving AI technologies between 2000 and 2016 show a positive and significant impact of AI patent families on employment. The effect, presumably of product innovations, is small in magnitude and limited to service sectors and younger firms, which are at the forefront of the leaders of the AI revolution. We also detect some evidence of increasing returns, suggesting that innovative companies more focused on AI technologies are achieving larger impacts in terms of job creation.

Transcriptomic Analysis of Mineralized Adipose-Derived Stem Cell Tissues for Calcific Valve Disease Modelling.

Calcific aortic valve disease (CAVD) is characterized by the fibrosis and mineralization of the aortic valve, which leads to aortic stenosis and heart failure. At the cellular level, this is due to the osteoblastic-like differentiation of valve interstitial cells (VICs), resulting in the calcification of the tissue. Unfortunately, human VICs are not readily available to study CAVD pathogenesis and the implicated mechanisms in vitro; however, adipose-derived stromal/stem cells (ASCs), carrying the patient's specific genomic features, have emerged as a promising cell source to model cardiovascular diseases due to their multipotent nature, availability, and patient-specific characteristics. In this study, we describe a comprehensive transcriptomic analysis of tissue-engineered, scaffold-free, ASC-embedded mineralized tissue sheets using bulk RNA sequencing. Bioinformatic and gene set enrichment analyses revealed the up-regulation of genes associated with the organization of the extracellular matrix (ECM), suggesting that the ECM could play a vital role in the enhanced mineralization observed in these tissue-engineered ASC-embedded sheets. Upon comparison with publicly available gene expression datasets from CAVD patients, striking similarities emerged regarding cardiovascular diseases and ECM functions, suggesting a potential link between ECM gene expression and CAVDs pathogenesis. A matrisome-related sub-analysis revealed the ECM microenvironment promotes the transcriptional activation of the master gene runt-related transcription factor 2 (RUNX2), which is essential in CAVD development. Tissue-engineered ASC-embedded sheets with enhanced mineralization could be a valuable tool for research and a promising avenue for the identification of more effective aortic valve replacement therapies.

Sleep Apnea is Not an Obstacle for Outpatient Hip and Knee Arthroplasty.

BACKGROUND: Up to 25% of patients requiring hip or knee arthroplasty have sleep apnea (SA), and these patients have historically been excluded from outpatient programs. The objectives of this study were to evaluate same-day discharge failure as well as 30-day complications, readmissions, and unexpected visits. METHODS: A retrospective case-control study comparing patients who have and do not have SA matched for age, sex and arthroplasty type (total hip arthroplasty, total knee arthroplasty, unicompartimental knee arthroplasty) who underwent primary outpatient surgery between February 2019 and December 2022 in 2 academic hospitals was conducted. Cases with mild SA, moderate SA with a body mass index (BMI) <35, and SA of all severity treated by continuous positive airway pressure machines were eligible. There were 156 patients included (78 cases). Complications were assessed according to the Clavien-Dindo Classification and the Comprehensive Complication Index. Continuous variables were evaluated by Student's T or Mann-Whitney tests, while categorical data were analyzed by Chi-square or Fisher tests. Univariate analyses were performed to determine discharge failure risk factors. RESULTS: There were 6 cases (7.7%) and 5 controls (6.4%) who failed to be discharged on surgery day (P = .754), with postoperative hypoxemia (6, [3.8%]) and apnea periods (3, [1.9%]) being the most common causes. Higher BMI (odds ratio = 1.19, P = .013) and general anesthesia (odds ratio = 11.97, P = .004) were found to be risk factors for discharge failure. No difference was observed on 30-day readmissions (P = .497), unexpected visits (P = 1.000), and complications on the Clavien-Dindo Classification (P > .269) and Comprehensive Complication Index (P > .334) scales. CONCLUSIONS: Selected patients who have SA can safely undergo outpatient hip or knee arthroplasty. Higher BMI and general anesthesia increased the odds of same-day discharge failure. LEVEL OF EVIDENCE: Level III, Case-control Study.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Antibiotics and Antibiotic Resistance-Mur Ligases as an Antibacterial Target.

The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic pathway represents opportunities for the development of novel antibacterial agents. Among the enzymes involved, Mur ligases, described herein, and especially the amide ligases MurC-F are key targets for the discovery of multi-inhibitors, as they share common active sites and structural features.

A fist bump in a political meeting? The influence of social context on affordance selection.

Historically, understanding human cognition such as action processing has been a challenging issue in cognitive neuropsychology and the more we know about cognition, the more we shape it as a complex, multi-determined phenomenon that is embedded in a social context. The present study aimed at understanding how the social context could influence affordance selection. We hypothesized that affordance selection would be modulated by social context and that a given hand configuration would be considered appropriate or not, as a function of the presence or absence of social context. Twenty-six healthy participants were asked to judge the appropriateness of three variants of 10 hand-object interactions based on photographs presented with or without a visual, social context. In our results, hand configurations were intrinsically acceptable or not, but this effect was modulated by the social context. A three-step model of the influence of social context on affordance selection was proposed, according to which selection depends on social norms, in the form of social knowledge and social context analysis.

Heterozygous NF1 dermal fibroblasts modulate exosomal content to promote angiogenesis in a tissue-engineered skin model of neurofibromatosis type-1.

Neovascularization is a critical process in tumor progression and malignant transformation associated with neurofibromatosis type 1 (NF1). Indeed, fibroblasts are known to play a key role in the tumoral microenvironment modification by producing an abundant collagenous matrix, but their contribution in paracrine communication pathways is poorly understood. Here, we hypothesized that NF1 heterozygosis in human dermal fibroblasts could promote angiogenesis through exosomes secretion. The purposes of this study are to identify the NF1 fibroblast-derived exosome protein contents and to determine their proangiogenic activity. Angiogenic proteome measurement confirmed the overexpression of VEGF and other proteins involved in vascularization. Tube formation of microvascular endothelial cells was also enhanced in presence of exosomes derived from NF1 skin fibroblasts. NF1 tissue-engineered skin (NF1-TES) generation showed a significantly denser microvessels networks compared to healthy controls. The reduction of exosomes production with an inhibitor treatment demonstrated a drastic decrease in blood vessel formation within the dermis. Our results suggest that NF1 haploinsufficiency alters the dermal fibroblast function and creates a pro-angiogenic signal via exosomes, which increases the capillary formation. This study highlights the potential of targeting exosome secretion and angiogenesis for therapeutic interventions in NF1.

Evaluation of the contribution of shotgun metagenomics in the microbiological diagnosis of liver abscesses.

BACKGROUND: Shotgun metagenomics (SMg) sequencing has gained a considerable interest, as it enables the detection of any microorganisms through a single analysis. Due to the limitations of standard microbiological approaches, the microbial documentation of liver abscesses (LA), which is crucial for their medical management, can be difficult. Here we aimed to compare the performance of SMg with standard approaches for the microbiological documentation of LA. METHODS: In this retrospective study conducted at two centers, we compared the results of standard microbiology with metagenomics analysis of consecutive LA samples. For samples tested positive for Klebsiella pneumoniae, we compared the analysis of virulence and resistance genes using metagenomics data to whole-genome sequencing of corresponding isolates obtained in culture. RESULTS: Out of the 62 samples included, standard approaches and SMg yielded documentation in 80.6% and 96.8%, respectively. In 37.1% (23/62) of cases, both methods showed identical results, whereas in 43.5% (27/62) of cases, the samples were positive by both methods, but SMg found additional species in 88.9% (24/27), mostly anaerobes. When the standard approaches were negative, the SMg was able to detect microorganisms in 80.0% of cases (8/10). Overall, SMg identified significantly more microorganisms than culture (414 vs.105; p<0.05). K. pneumoniae genome analysis was able to detect resistance and virulence genes with a level of sensitivity depending on the depth of sequencing. DISCUSSION: Overall, we showed that SMg had better performance in detecting and identifying microorganisms from LA samples and could help characterizing strain's resistome and virulome. Although still costly and requiring specific skills and expensive equipment, MGs methods are set to expand in the future.

Grip selection without tool knowledge: end-state comfort effect in familiar and novel tool use.

A well-known phenomenon for the study of movement planning is the end-state comfort (ESC) effect: When they reach and grasp tools, individuals tend to adopt uncomfortable initial hand postures if that allows a subsequent comfortable final posture. In the context of tool use, this effect is modulated by tool orientation, task goal, and cooperation. However, the cognitive bases of the ESC effect remain unclear. The goal of this study was to determine the contribution of semantic tool knowledge and technical reasoning to movement planning, by testing whether the ESC effect typically observed with familiar tools would also be observed with novel tools. Twenty-six participants were asked to reach and grasp familiar and novel tools under varying conditions (i.e., tool's handle downward vs. upward; tool transport vs. use; solo vs. cooperation). In our findings, the effects of tool orientation, task goal and cooperation were replicated with novel tools. It follows that semantic tool knowledge is not critical for the ESC effect to occur. In fact, we found an "habitual" effect: Participant adopted uncomfortable grips with familiar tools even when it was not necessary (i.e., to transport them), probably because of the interference of habitual movement programming with actual movement programming. A cognitive view of movement planning is proposed, according to which goal comprehension (1) may rely on semantic tool knowledge, technical reasoning, and/or social skills, (2) defines end-state configuration, which in turn (3) calibrates beginning-state comfort and hence the occurrence of the ESC effect.

Spherical rotary cell seeding system for production of small-caliber tissue-engineered blood vessels with complex geometry.

Entirely biological human tissue-engineered blood vessels (TEBV) were previously developed for clinical use. Tissue-engineered models have also proven to be valuable tools in disease modelling. Moreover, there is a need for complex geometry TEBV for study of multifactorial vascular pathologies, such as intracranial aneurysms. The main goal of the work reported in this article was to produce an entirely human branched small-caliber TEBV. The use of a novel spherical rotary cell seeding system allows effective and uniform dynamic cell seeding for a viable in vitro tissue-engineered model. In this report, the design and fabrication of an innovative seeding system with random spherical 360° rotation is described. Custom made seeding chambers are placed inside the system and hold Y-shaped polyethylene terephthalate glycol (PETG) scaffolds. The seeding conditions, such as cell concentration, seeding speed and incubation time were optimized via count of cells adhered on the PETG scaffolds. This spheric seeding method was compared to other approaches, such as dynamic and static seeding, and clearly shows uniform cell distribution on PETG scaffolds. With this simple to use spherical system, fully biological branched TEBV constructs were also produced by seeding human fibroblasts directly on custom-made complex geometry PETG mandrels. The production of patient-derived small-caliber TEBVs with complex geometry and optimized cellular distribution all along the vascular reconstructed may be an innovative way to model various vascular diseases such as intracranial aneurysms.

Tridimensional cell culture of dermal fibroblasts promotes exosome-mediated secretion of extracellular matrix proteins.

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis's ECM.

Synthesis and Structure-Activity Relationship Studies of Pyrido [1,2-e]Purine-2,4(1H,3H)-Dione Derivatives Targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis.

In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 µM) on PBM cells.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells.

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Tunable Approach to C-Linked Analogs of Glycosamines.

The synthesis of (1R)-2-amino-2-deoxy-ß-l-gulopyranosyl benzene and the α and ß forms of 2-amino-2-deoxy-l-idopyranosyl benzene derivatives was accomplished through stereospecific addition of tributylstannyllithium to readily available (SR)- or (SS)-N-tert-butanesulfinyl-arabinofuranosylamine building blocks, followed by stereoretentive Pd-catalyzed Migita-Kosugi-Stille cross-coupling, stereoselective reduction, and an activation-cyclization strategy. Application of this methodology paves the way to new three-dimensional chemical space and preparation of unknown (non-natural) and complex 2-amino-2-deoxy sugars of biological interest.

Nucleosides and emerging viruses: A new story.

With several US Food and Drug Administration (FDA)-approved drugs and high barriers to resistance, nucleoside and nucleotide analogs remain the cornerstone of antiviral therapies for not only herpesviruses, but also HIV and hepatitis viruses (B and C); however, with the exception of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which vaccines have been developed at unprecedented speed, there are no vaccines or small antivirals yet available for (re)emerging viruses, which are primarily RNA viruses. Thus, herein, we present an overview of ribonucleoside analogs recently developed and acting as inhibitors of the viral RNA-dependent RNA polymerase (RdRp). They are new lead structures that will be exploited for the discovery of new antiviral nucleosides.

Chemical Approaches to Carbocyclic Nucleosides.

Nucleoside analogues are at the forefront of antiviral therapy for last decades. To circumvent some of their limitations, based on their metabolism, and in order to improve their anti-viral potency and selectivity, several families of nucleoside analogues have been described through structural modifications at the sugar and heterocycles. The replacement of the oxygen of the nucleoside by a methylene has led to the family of carbocyclic (or cyclopentane) nucleoside analogues. Various potent anti-HIV and anti-HBV drugs belong to this family. Main syntheses of carbocyclic analogues of nucleosides used Diels-Alder reactions (in racemic or asymmetric series), but also started from carbohydrates (ribose, glucose), as a source of optically active compounds, which then had to be transformed into carbacycles under various conditions. The growing interest in carbocyclic nucleosides has led several groups, including ours, to develop new analogues and to explore novel routes. This article will review some of the recent chemistry developed on selected five-membered ring carbocyclic nucleosides.

Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.

BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

RNF213 Loss-of-Function Promotes Angiogenesis of Cerebral Microvascular Endothelial Cells in a Cellular State Dependent Manner.

Enhanced and aberrant angiogenesis is one of the main features of Moyamoya disease (MMD) pathogenesis. The ring finger protein 213 (RNF213) and the variant p.R4810K have been linked with higher risks of MMD and intracranial arterial occlusion development in east Asian populations. The role of RNF213 in diverse aspects of the angiogenic process, such as proliferation, migration and capillary-like formation, is well-known but has been difficult to model in vitro. To evaluate the effect of the RNF213 MMD-associated gene on the angiogenic activity, we have generated RNF213 knockout in human cerebral microvascular endothelial cells (hCMEC/D3-RNF213-/-) using the CRISPR-Cas9 system. Matrigel-based assay and a tri-dimensional (3D) vascularized model using the self-assembly approach of tissue engineering were used to assess the formation of capillary-like structures. Quite interestingly, this innovative in vitro model of MMD recapitulated, for the first time, disease-associated pathophysiological features such as significant increase in angiogenesis in confluent endothelial cells devoid of RNF213 expression. These cells, grown to confluence, also showed a pro-angiogenic signature, i.e., increased secretion of soluble pro-angiogenic factors, that could be eventually used as biomarkers. Interestingly, we demonstrated that that these MMD-associated phenotypes are dependent of the cellular state, as only noted in confluent cells and not in proliferative RNF213-deficient cells.