Early Steps Research Articles

Fast mobility analysis for generative design applications through a real vector space representation of mobilities

During the early stages of design, mechanisms are commonly modeled as perfect joints assembled with infinitely rigid bodies. This representation enables the prediction of the system’s mobilities through a mobility analysis. However, traditional mobility analysis tools can be computationally expensive or lack critical information, such as the type or direction of mobilities. It hinders the generation of topology and configuration through generative design schemes.In this paper, we propose an alternative approach to mobility analysis based on a real vector space representation of mobilities. Our method provides relevant information for early design steps while being computationally effective through a novel formulation of series and parallel assembly topological operations. A benchmark on four selected use cases highlights an acceleration of 3 to 4 orders of magnitude compared to traditional approaches. Additionally, design rules on the joints’ positions can be automatically generated with our approach. It enables the automation of the complete design process, including topology and configuration. As such, we provide guidelines to develop a generative design scheme dedicated to the synthesis of guiding mechanisms.

Flagellar point mutation causes social aggregation in laboratory-adapted Bacillus subtilis under conditions that promote swimming.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering.

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 Å. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Heterotypic interaction promotes asymmetric division of human hematopoietic progenitors.

Hematopoietic stem and progenitor cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes, including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interactions with osteoblast and endothelial cells promote asymmetric divisions of human HSPCs. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of the differentiation marker CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.

Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production.

Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimer's disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid β-peptides (Aβ) by the PSEN1-containing γ-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by γ-secretase is processive, involving initial endoproteolysis to produce long Aβ peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled γ-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a C. elegans model of FAD independently of Aβ production. Here we conducted full quantitative analysis of all proteolytic events on APP substrate by γ-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled γ-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in Aβ-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of Aβ intermediates. These findings provide further evidence that FAD mutations lead stalled and stabilized γ-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of γ-secretase proteolysis as the pathogenic trigger.

Effect of Spheroidization and Inoculation on the Early Solidification Steps of Hypereutectic Cast Irons

Effect of Spheroidization and Inoculation on the Early Solidification Steps of Hypereutectic Cast Irons

A20 haploinsufficiency disturbs immune homeostasis and drives the transformation of lymphocytes with permissive antigen receptors.

Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 34 patients with HA20, we show that heterozygous TNFAIP3 loss skews immune repertoires toward lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas. This skewing was mediated by a feed-forward tumor necrosis factor (TNF)/A20/nuclear factor κB (NF-κB) loop that shaped pre-lymphoma transcriptome signatures in clonally expanded B (CD81, BACH2, and NEAT1) or T (GATA3, TOX, and PDCD1) cells. The skewing was reversed by anti-TNF treatment but could also progress to overt lymphoma. Analysis of conditional TNFAIP3 knock-out mice reproduced the wiring of the TNF/A20/NF-κB signaling axis with permissive antigen receptors and suggested a distinct regulation in B and T cells. Together, patients with the genetic disorder HA20 provide an exceptional window into A20/TNF/NF-κB-mediated control of immune homeostasis and early steps of lymphomagenesis that remain clinically unrecognized.

High Expression of AhR and Environmental Pollution as AhR-Linked Ligands Impact on Oncogenic Signaling Pathways in Western Patients with Gastric Cancer-A Pilot Study.

The vast majority of gastric cancer (GC) cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GC, often associated with poor overall survival, has constantly increased in Western countries. Epidemiological studies have reported increased mortality from GC after occupational exposure to pro-carcinogens that are metabolically activated by cytochrome P450 enzymes through aryl hydrocarbon receptor (AhR). However, little is known about the role of AhR and environmental AhR ligands in diffuse GC as compared to intestinal GC in Western patients. In a cohort of 29, we demonstrated a significant increase in AhR protein and mRNA expression levels in GCs independently of their subtypes and clinical parameters. AhR and RHOA mRNA expression were correlated in diffuse GC. Further, our study aimed to characterize in GC how AhR and the AhR-related genes cytochrome P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) affect the mRNA expression of a panel of genes involved in cancer development and progression. In diffuse GC, CYP1A1 expression correlated with genes involved in IGF signaling, epithelial-mesenchymal transition (Vimentin), and migration (MMP2). Using the poorly differentiated KATO III epithelial cell line, two well-known AhR pollutant ligands, namely 2-3-7-8 tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (BaP), strongly increased the expression of CYP1A1 and Interleukin1β (IL1B), and to a lesser extend UGT1, NQO1, and AhR Repressor (AhRR). Moreover, the increased expression of CYP1B1 was seen in diffuse GC, and IHC staining indicated that CYP1B1 is mainly expressed in stromal cells. TCDD treatment increased CYP1B1 expression in KATO III cells, although at lower levels as compared to CYP1A1. In intestinal GC, CYP1B1 expression is inversely correlated with several cancer-related genes such as IDO1, a gene involved in the early steps of tryptophan metabolism that contributes to the endogenous AhR ligand kynurenine expression. Altogether, our data provide evidence for a major role of AhR in GC, as an environmental xenobiotic receptor, through different mechanisms and pathways in diffuse and intestinal GC. Our results support the continued efforts to clarify the identity of exogenous AhR ligands in diffuse GC in order to define new therapeutic strategies.

Antiviral Efficacy of Coridothymus capitatus Essential Oil Against HSV-1 and HSV-2.

Coridothymus capitatus is a perennial herb with aromatic leaves and flowers, distinct from Thymus vulgaris in its chemical composition, resulting in a unique Thymus Essential Oil (TEO). A main component of TEO, carvacrol, is known for its antimicrobial and insecticidal activity. Carvacrol has potent antibacterial, antioxidant, anti-inflammatory, and antifungal properties, generating interest in traditional medicine. However, studies on its antiviral activity are limited. Given the rise in viral infections and limitations of synthetic antiviral drugs, natural antiviral agents are promising due to their efficacy, lower resistance development, and reduced side effects. This study assessed the antiviral efficacy of TEO compared to that of pure carvacrol. We tested various viruses, revealing significant inhibitory effects of TEO on the replication of only Simplexvirus humanalpha1 (HSV-1) and Simplexvirus humanalpha2 (HSV-2), with specific interference during the early stages of the viral replication cycle after the adsorption period. TEO exhibited inhibitory effects at doses below the cytotoxic threshold, with IC50 values of 47 μg/mL for HSV-1 and 40 μg/mL for HSV-2. Maximum virus inhibition was achieved when TEO was added within 90 min post-infection, indicating interference with early viral replication steps. These findings highlight the potential of TEO as a natural antiviral agent and suggest further research into its mechanisms and clinical applications.

Examining the Complex Mismatch Negativity in Early Phase Psychosis Using the Dual Rule Paradigm.

Using electroencephalography (EEG) to examine the simple mismatch negativity (MMN), a marker of auditory cortex function, has been of great interest in the exploration of biomarkers for psychotic illness. Despite many studies reporting MMN deficits in chronic schizophrenia, there are inconsistent reports of MMN reductions in the early phases of psychotic illness, suggesting the MMN elicited by traditional paradigms may not be a sensitive enough measure of vulnerability to be used as a biomarker. Recently, a more computationally complex measure of auditory cortex function (the complex mismatch negativity; cMMN) has been hypothesized to provide a more sensitive marker of illness vulnerability. The current study employed a novel dual rule paradigm, in which two pattern rules are established and violated, to examine the cMMN in 14 individuals with early phase psychosis (EPP, < 5 years illness) and 15 healthy controls (HC). Relationships between cMMN waveforms, symptom severity, and measures of functioning were explored. We found reductions of cMMN amplitudes at the site of maximal amplitude in EPP (p = .017) with large effect sizes (Hedges' g = 0.96). This study is an early step in the exploration of the cMMN as a biomarker for psychosis. Our results provide evidence that the dual rule cMMN paradigm shows promise as a method for cMMN elicitation that captures more subtle neurofunctional changes in the early stages of illness.

Increased vascular deposition of oxidized LDL in untreated juvenile dermatomyositis

BackgroundJuvenile dermatomyositis (JDM) is a systemic vasculopathy associated with metabolic derangements and possible increased risk for premature atherosclerosis. Oxidation of low-density lipoprotein (LDL) in the endothelium is an early step in atherosclerotic plaque formation. It is not known if oxidized LDL is altered in children with untreated JDM. The deposition of oxidized LDL in the vasculature of muscle biopsies (MBx) from patients with untreated JDM and pediatric controls was assessed.FindingsFrozen tissue sections of MRI-directed MBx from 20 female children with untreated JDM and 5 female controls were stained with DAPI and fluorescently labeled antibodies against von Willebrand factor (vWF) and LDL oxidized by copper (oxLDL). Blood vessels were identified by positive vWF staining, and total fluorescence of oxLDL within the vessel walls was measured. Children with untreated JDM had increased deposition of oxLDL in the walls of muscle vasculature compared to healthy children (difference in means ± SEM = 19.86 ± 8.195, p = 0.03). Within the JDM cohort, there was a trend towards increased oxLDL deposition with longer duration of untreated disease (r = 0.43, p = 0.06). There was no significant correlation found between oxLDL deposition and markers of acute JDM disease activity including disease activity scores or muscle enzymes.ConclusionsThis study found increased deposition of oxLDL within blood vessels of children with untreated JDM supporting the concern that these children are at increased risk for premature atherosclerosis from chronic exposure to vascular oxLDL. This study highlights the importance of early diagnosis and treatment initiation to ameliorate cardiovascular damage.

Quinolinonyl Derivatives as Dual Inhibitors of the HIV-1 Integrase Catalytic Site and Integrase-RNA interactions.

The HIV-1 integrase (IN) plays a critical role in the viral lifecycle by integrating the viral DNA into the host chromosome. The catalytic function of IN has been exploited as a target, with five drugs acting as active site binders (IN strand transfer inhibitors, INSTIs). However, IN mutations conferring low-level resistance to INSTIs have been reported. Therefore, new IN inhibitors with different mechanisms of action are needed. The allosteric inhibition of IN, exerted by allosteric IN inhibitors (ALLINIs), is gaining interest. ALLINIs inhibit IN by inducing aberrant IN multimerization with different mechanisms. Furthermore, recent discoveries unveiled that IN has an under-studied yet equally vital second function. This involves IN binding to the RNA genome in virions, necessary for proper virion maturation. In this work, we describe a series of quinolinonyl derivatives as inhibitors of both the IN catalytic functions and IN-RNA interactions, which impair both early and late steps of viral replication.

The use of conceptual ecological models to identify critical data and uncertainties to support numerical modeling: The northern Gulf of Mexico eastern oyster Crassostrea virginica example

AbstractObjectiveIncreasing reliance on numerical simulation models to help inform management and restoration choices benefits from careful consideration of critical early steps in model development. Along the northern coast of the Gulf of Mexico, the eastern oyster Crassostrea virginica fulfills important ecological and economic roles. Using the eastern oyster as an example, we draw on several recent frameworks outlining best practices for model development and application for restoration, conservation, and management.MethodsWe identify priority model questions, outline a conceptual ecological model (CEM) to guide numerical model development, and use this framework to identify uncertainties and research needs.ResultThe CEM uses a nested design, identifying explicit vital rates, processes, attributes, and outcomes for the species (oysters), population, and metapopulation (i.e., network of populations) levels in response to drivers of species, population, and metapopulation changes and changing environmental factors. Most management actions related to oyster restoration and harvest affect population attributes directly, but many coastal management actions and changes (i.e., climate change and coastal and water resource engineering) impact environmental factors that alter vital rates and attributes of oysters, populations, and metapopulations.ConclusionInvestment in studies targeting individual oyster‐ and population‐level multi‐stressor responses (filtration, respiration, growth, and reproduction) and improving hydrodynamic and environmental models targeting drivers that influence metapopulation vital rates and attributes (i.e., connectivity and substrate persistence) would contribute to reducing uncertainties. Development of numerical models covering the entire oyster life cycle and connectivity of populations using hydrodynamic models of current and predicted conditions to provide key abiotic and biotic factors influencing larval movement, recruitment, and on‐reef oyster vital rates would assist in balancing the goals of conservation, restoration, and fisheries management of this foundational estuarine species.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

Poly(rC)-Binding Protein 2 Does Not Directly Participate in HCV Translation or Replication, but Rather Modulates Genome Packaging.

The hepatitis C virus (HCV) co-opts many cellular factors-including proteins and microRNAs-to complete its life cycle. A cellular RNA-binding protein, poly(rC)-binding protein 2 (PCBP2), was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, using the HCV cell culture (HCVcc) system and assays that isolate each step of the viral life cycle, we found that PCBP2 does not have a direct role in viral entry, translation, genome stability, or HCV RNA replication. Rather, our data suggest that PCBP2 depletion only impacts viral RNAs that can undergo genome packaging. Taken together, our data suggest that endogenous PCBP2 modulates the early steps of genome packaging, and therefore only has an indirect effect on viral translation and RNA replication, likely by increasing the translating/replicating pool of viral RNAs to the detriment of virion assembly.

Metastatic breast cancer cells are metabolically reprogrammed to maintain redox homeostasis during metastasis

Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2′-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations.Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.

Numb-associated kinases regulate sandfly-borne Toscana virus entry

ABSTRACT Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited. This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle.

Adaptation of Saccharomyces to High Glucose Concentrations and Its Impact on Growth Kinetics of Alcoholic Fermentations.

Prior adaptation of Saccharomyces cerevisiae to the fermentation medium ensures its implantation and success in alcoholic fermentations. Fermentation kinetics can be characterized with mathematical models to objectively measure the success of adaptation and growth. The study aims at assessing and comparing two pre-culture procedures using, respectively, one or two adaptation steps, analyzing the impact of different initial glucose concentrations on the fermentation profiles of S. cerevisiae cultures, and assessing the performance of three predictive growth models (Buchanan's, modified Gompertz, and Baranyi and Roberts models) under varied initial glucose concentrations. We concluded that both protocols produced S. cerevisiae pre-cultures with similar viability and biomass increase, which suggests that short protocols may be more cost-effective. Furthermore, the study highlights the need of inoculating a high S. cerevisiae population to minimize the depletion of dissolved oxygen in the medium and to ensure that glucose is predominantly directed toward the ethanol formation at early fermentative steps. This study shows that the relationship between kinetic parameters is model-dependent, which hinders inter-study comparisons and stresses the need for standardized growth models. We advocate for the generalized use of confidence intervals of the kinetic parameters to facilitate objective inter-study comparisons.