A new microscopy pipeline for studying the initial stages of nuclear and micronuclear rupture and repair.

Nuclear envelope repair is a fundamental cellular response to stress, especially for cells experiencing frequent nuclear ruptures, such as cancer cells. Moreover, for chromosomally unstable cancer cells, characterized by the presence of micronuclei, the irreversible rupture of these structures constitutes a fundamental step toward cancer progression and therapy resistance. For these reasons, the study of nuclear envelope rupture and repair is of paramount importance. Nonetheless, due to the constraint imposed by the stochastic nature of rupture events, a precise characterization of the initial stage of nuclear repair remains elusive. In this study, we overcame this limitation by developing a new imaging pipeline that deterministically induces rupture while simultaneously imaging fluorescently tagged repair proteins. We provide a detailed step-by-step protocol to implement this method on any confocal microscope and applied it to study the major nuclear repair protein, barrier-to-autointegration factor (BAF). As a proof of principle, we demonstrated two different downstream analysis methods and showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment at primary nuclei of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and Charged Multivesicular Protein 7 (CHMP7), the scaffolding protein of the endosomal sorting complex required for transport III (ESCRT-III) membrane remodeling complex. The CHMP7-LEMD2 binding is the fundamental step allowing the recruitment of ESCRT-III, which represents the other major nuclear repair mechanism. This demonstrates the method's applicability for investigating protein dynamics at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair.

Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.

KEY MESSAGE: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

BACKGROUND: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis.

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

Introduction: HIV-1 envelope (Env) is the key target for antibodies (Abs) against the virus and thus an important HIV-1 vaccine component. Env is synthesized from a gp160 precursor with a signal peptide (SP) at its N-terminus. This study investigated the influence of the SP on Env antigenicity and immunogenicity. Methods: Env proteins from two HIV-1 isolates, AA05 and AC02, were analyzed as gp120 and gp160 in their native wild-type (WT) forms and as chimeras with swapped SPs (AA05-02 and AC02-05). The WT and chimeric Env were assessed for antigenicity and glycosylation using monoclonal antibodies (mAbs) and glycan probes. Immunogenicity was tested in mice using three vaccine types: gp120 protein, gp120 DNA+gp120 protein, and gp120 DNA+gp160 DNA. Results: The recombinant AC02 gp120 protein was antigenically superior to AA05 as indicated by higher reactivity with most mAbs tested. When SPs were swapped, the antigenicity of the chimeric gp120s (AA05-02 and AC02-05) resembled that of the gp120s from which the SPs were derived; AA05-02 was similar to AC02 and vice versa. Glycan probe reactivity followed a similar pattern: AA05-02 and AC02 showed similar affinity to high-mannose specific mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02. Conclusion: These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines.

CHK1 controls zygote pronuclear envelope breakdown by regulating F-actin through interacting with MICAL3.

CHK1 mutations could cause human zygote arrest at the pronuclei stage, a phenomenon that is not well understood at the molecular level. In this study, we conducted experiments where pre-pronuclei from zygotes with CHK1 mutation were transferred into the cytoplasm of normal enucleated fertilized eggs. This approach rescued the zygote arrest caused by the mutation, resulting in the production of a high-quality blastocyst. This suggests that CHK1 dysfunction primarily disrupts crucial biological processes occurring in the cytoplasm. Further investigation reveals that CHK1 mutants have an impact on the F-actin meshwork, leading to disturbances in pronuclear envelope breakdown. Through co-immunoprecipitation and mass spectrometry analysis of around 6000 mouse zygotes, we identified an interaction between CHK1 and MICAL3, a key regulator of F-actin disassembly. The gain-of-function mutants of CHK1 enhance their interaction with MICAL3 and increase MICAL3 enzymatic activity, resulting in excessive depolymerization of F-actin. These findings shed light on the regulatory mechanism behind pronuclear envelope breakdown during the transition from meiosis to the first mitosis in mammals.

Mechanisms for assembly of the nucleoplasmic reticulum.

The nuclear envelope consists of an outer membrane connected to the endoplasmic reticulum, an inner membrane facing the nucleoplasm and a perinuclear space separating the two bilayers. The inner and outer nuclear membranes are physically connected at nuclear pore complexes that mediate selective communication and transfer of materials between the cytoplasm and nucleus. The spherical shape of the nuclear envelope is maintained by counterbalancing internal and external forces applied by cyto- and nucleo-skeletal networks, and the nuclear lamina and chromatin that underly the inner nuclear membrane. Despite its apparent rigidity, the nuclear envelope can invaginate to form an intranuclear membrane network termed the nucleoplasmic reticulum (NR) consisting of Type-I NR contiguous with the inner nuclear membrane and Type-II NR containing both the inner and outer nuclear membranes. The NR extends deep into the nuclear interior potentially facilitating communication and exchanges between the nuclear interior and the cytoplasm. This review details the evidence that NR intrusions that regulate cytoplasmic communication and genome maintenance are the result of a dynamic interplay between membrane biogenesis and remodelling, and physical forces exerted on the nuclear lamina derived from the cyto- and nucleo-skeletal networks.

Unveiling the roles of LEMD proteins in cellular processes.

Proteins localized in the inner nuclear membrane (INM) engage in various fundamental cellular processes via their interactions with outer nuclear membrane (ONM) proteins and nuclear lamina. LAP2-emerin-MAN1 domain (LEMD) family proteins, predominantly positioned in the INM, participate in the maintenance of INM functions, including the reconstruction of the nuclear envelope during mitosis, mechanotransduction, and gene transcriptional modulation. Malfunction of LEMD proteins leads to severe tissue-restricted diseases, which may manifest as fatal deformities and defects. In this review, we summarize the significant roles of LEMD proteins in cellular processes, explains the mechanisms of LEMD protein-related diseases, and puts forward questions in less-explored areas like details in tissue-restricted phenotypes. It intends to sort out previous works about LEMD proteins and pave way for future researchers who might discover deeper mechanisms of and better treatment strategies for LEMD protein-related diseases.

Tail-anchored membrane protein SLMAP3 is essential for targeting centrosomal proteins to the nuclear envelope in skeletal myogenesis.

The positioning and communication between the nucleus and centrosomes are essential in cell division, differentiation and tissue formation. During skeletal myogenesis, the nuclei become evenly spaced with the switch of the microtubule-organizing centre (MTOC) from the centrosome to the nuclear envelope (NE). We report that the tail-anchored sarcolemmal membrane associated protein 3 (SLMAP3), a component of the MTOC and NE, is crucial for myogenesis because its deletion in mice leads to a reduction in the NE-MTOC formation, mislocalization of the nuclei, dysregulation of the myogenic programme and abnormal embryonic myofibres. SLMAP3-/- myoblasts also displayed a similar disorganized distribution of nuclei with an aberrant NE-MTOC and defective myofibre formation and differentiation programming. We identified novel interactors of SLMAP3, including pericentrin, PCM1 (pericentriolar material 1), AKAP9 (A-kinase anchoring protein 9), kinesin-1 members Kif5B (kinesin family member 5B), KCL1 (kinesin light chain 1), KLC2 (kinesin light chain 2) and nuclear lamins, and observed that the distribution of centrosomal proteins at the NE together with Nesprin-1 was significantly altered by the loss of SLMAP3 in differentiating myoblasts. SLMAP3 is believed to negatively regulate Hippo signalling, but its loss was without impact on this pathway in developing muscle. These results reveal that SLMAP3 is essential for skeletal myogenesis through unique mechanisms involving the positioning of nuclei, NE-MTOC dynamics and gene programming.

Dose-escalation, tolerability, and efficacy of intratumoral and subcutaneous injection of hemagglutinating virus of Japan envelope (HVJ-E) against chemotherapy-resistant malignant pleural mesothelioma: a clinical trial.

The hemagglutinating virus of Japan envelope (HVJ-E) is an inactivated Sendai virus particle with antitumor effect and inducing antitumor immunity. However, its dosage and efficacy have not been verified. We conducted a phase I clinical study on chemotherapy-resistant malignant pleural mesothelioma (MPM) aiming to determine the recommended dosage for a phase II study through dose-limiting toxicity and evaluate HVJ-E's preliminary efficacy. HVJ-E was administered intratumorally and subcutaneously to the patients with chemotherapy-resistant MPM. While no serious adverse events occurred, known adverse events of HVJ-E were observed. In the preliminary antitumor efficacy using modified response evaluation criteria in solid tumors (RECIST) criteria, three low-dose patients exhibited progressive disease, while all high-dose patients achieved stable disease, yielding disease control rates (DCRs) of 0% and 100%, respectively. Furthermore, the dose-dependent effect of HVJ-E revealed on DCR modified by RECIST and the baseline changes in target lesion size (by CT and SUL-peak; p < 0.05). Comparing targeted lesions receiving intratumoral HVJ-E with non-injected ones, while no clear difference existed at the end of the study, follow-up cases suggested stronger antitumor effects with intratumoral administration. Our findings suggest that HVJ-E could be safely administered to patients with chemotherapy-resistant MPM at both study doses. HVJ-E exhibited some antitumor activity against chemotherapy-resistant MPM, and higher doses tended to have stronger antitumor effects than lower doses. Consequently, a phase II clinical trial with higher HVJ-E doses has been conducted for MPM treatment. Trial registration number: UMIN Clinical Trials Registry (#UMIN000019345).

Insight into the outer membrane asymmetry of P. aeruginosa and the role of MlaA in modulating the lipidic composition, mechanical, biophysical, and functional membrane properties of the cell envelope.

In Gram-negative bacteria, the outer membrane (OM) is asymmetric, with lipopolysaccharides (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet. The asymmetry is maintained by the Mla system (MlaA-MlaBCDEF), which contributes to lipid homeostasis by removing mislocalized GPLs from the outer leaflet of the OM. Here, we ascribed how Pseudomonas aeruginosa ATCC 27853 coordinately regulates pathways to provide defense against the threats posed by the deletion of mlaA. Especially, we explored (i) the effects on membrane lipid composition including LPS, GPLs, and lysophospholipids, (ii) the biophysical properties of the OM such as stiffness and fluidity, and (iii) the impact of these changes on permeability, antibiotic susceptibility, and membrane vesicles (MVs) generation. Deletion of mlaA induced an increase in total GPLs and a decrease in LPS level while also triggering alterations in lipid A structures (arabinosylation and palmitoylation), likely to be induced by a two-component system (PhoPQ-PmrAB). Altered lipid composition may serve a physiological purpose in regulating the mechanobiological and functional properties of P. aeruginosa. We demonstrated an increase in cell stiffness without alteration of turgor pressure and inner membrane (IM) fluidity in ∆mlaA. In addition, membrane vesiculation increased without any change in OM/IM permeability. An amphiphilic aminoglycoside derivative (3',6-dinonyl neamine) that targets P. aeruginosa membranes induced an opposite effect on ∆mlaA strain with a trend toward a return to the situation observed for the WT strain. Efforts dedicated to understanding the crosstalk between the OM lipid composition, and the mechanical behavior of bacterial envelope, is one needed step for designing new targets or new drugs to fight P. aeruginosa infections.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium responsible for severe hospital-acquired infections. The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore, compromising this structure could increase sensitivity to antibiotics. The OM is asymmetric with the highly packed lipopolysaccharide monolayer at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. In this study, we show that deleting mlaA, the membrane component of Mla system located at the OM, affects the mechanical and functional properties of P. aeruginosa cell envelope. Our results provide insights into the role of MlaA, involved in the Mla transport pathway in P. aeruginosa.

Preliminary investigation and analysis of nucleotide site variability of nine glycoproteins on varicella-zoster virus envelope, Jilin Province, China, 2010-March 2024.

Varicella is endemic worldwide. In China, varicella has not yet been included in the list of legal infectious diseases, nor has a unified national surveillance program been established. And the live attenuated varicella vaccine has not been included in routine immunization. In this study, we analyzed for the first time the varicella epidemiology in Jilin Province in the past 20 years, and the nucleotide site, amino acid site and N-glycosylation site variation of glycoprotein in varicella-zoster virus (VZV) surface 9 in the past 15 years. The results showed that the reported incidence of varicella in Jilin Province in the last 20 years was fluctuating above and below 20/100,000, especially after the epidemic of the COVID-19, and fatal cases appeared in individual years. The genotypic branching of VZV was monitored as Clade 2 in the last 15 years. 9 glycogen nucleotide sites of VZV had different degrees of variability, and the variability had specificity. Therefore, it gives us the idea that in order to reduce the incidence of varicella and herpes zoster, a provincial or even national surveillance program should be introduced as early as possible, and the dynamic monitoring of the variability of the nucleotide sites of VZV should be strengthened at the same time as the vaccine immunization strategy is introduced.

High-resolution 2D solid-state NMR provides insights into nontuberculous mycobacteria.

We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Hydrated NTM samples were studied at natural abundance without isotope-labelling, as whole-cells versus cell envelope isolates, and native versus fixed sample preparations. We utilized 1D13C and 2D 1H-13C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than ∼100 distinct 13C signals were identified in the ssNMR spectra. We provide tentative assignments for ∼30 polysaccharides by using well resolved 1H/13C chemical shifts from the 2D INEPT-based 1H-13C ssNMR spectrum. The signals originating from both the flexible and rigid fractions of the whole-cell bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. The majority of the 13C signals were not affected by fixation of the whole cell samples. The isolated cell envelope NMR spectrum overlap with the whole-cell spectrum to a large extent, where the latter has more signals. As an orthogonal way of characterizing these bacteria, electron microscopy (EM) was used to provide spatial information. ssNMR and EM data suggest that the M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize NTM strains and identify chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.

Selection of DNA aptamers against Chikungunya virus Envelope 2 Protein and their application in sandwich ELASA.

Chikungunya fever, caused by Chikungunya virus (CHIKV) exhibits clinical features that mimic that of other arbovirus infections such as dengue. CHIKV Envelope 2 (E2) protein, an antigenic epitope of CHIKV, has been identified as an ideal marker for diagnostics. The current CHIKV antigen detection tests are largely based on antibodies but are beleaguered by issues such as sensitivity to high temperature, expensive and prone to batch-to-batch variations. Aptamers are suitable alternatives to antibodies as they are cheaper and have no batch-to-batch variations compared to antibodies. In this study, DNA aptamer selection against CHIKV E2 proteins was performed using two different randomized ssDNA libraries. Chik-2 (96-mer) and Chik-3 (76-mer) were isolated from these two libraries and were identified as the potential aptamers against CHIKV E2 protein. The binding affinity of Chik-2 and Chik-3 against CHIKV E2 protein was estimated at 177.5 ± 32.69 nM and 30.01 ± 3.60 nM, respectively. A sandwich ELASA was developed, and this assay showed a detection limit of 2.17 x 103 PFU/mL. The sensitivity and specificity of the assay were 80 % and 100 %, respectively. The assay showed no cross-reactivity with dengue-positive samples, demonstrating the enormous diagnostic potential of these aptamers for the detection of CHIKV.

Data-Driven and Cell-Specific Determination of Nuclei-Associated Actin Structure.

Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F-actin.

Envelope domain III E324, E351, and E380 mutations lever adaptive evolution of DENV-1 genotype I.

Dengue virus (DENV) gains genetic mutations during continuous transmission and evolution, making the virus more adaptive and virulent. The clade of DENV-1 genotype I has expanded and become the predominant genotype in Asia and the Pacific areas, but the underlying mechanisms are unclear. A combined analysis of nonsynonymous mutations in domain III of the envelope protein and their biological effects on virus pathogenesis and transmission was evaluated. Phylogenetic analyses found three nonsynonymous mutations (V324I, V351L, and V380I) in domain III of the envelope protein, which emerged in 1970s-1990s and stably inherited and expanded in contemporary strains after 2000. We generated reverse-mutated viruses (I324V, L351V, and I380V) based on an infectious clone of an epidemic DENV-1 strain (NIID02-20), and the results suggested that the infectivity of the contemporary epidemic virus (wild type, WT) has increased compared to the reverse mutant viruses in mammalian hosts but not mosquito vectors. The WT virus showed a higher binding affinity to host cells and increased virion stability. In addition, weaker immunogenicity and higher resistance to neutralizing antibodies of the WT virus indicated a trend of immune escape. The data suggested that nonsynonymous mutations of the E protein (V324I, V351L, and V380I) promote infectivity and immune evasion of DENV-1 genotype I, which may facilitate its onward transmission on a global scale. IMPORTANCE: We provide evidence that minor sequence variation among dengue virus (DENV) strains can result in increased adaptability and virulence, impacting both the biology of the virus and the antiviral immune response. The genetic mutations of DENV-1 gained during continuous transmission and evolution will offer new clues for the design of novel vaccines against flaviviruses.

Ultrasound attenuation imaging as a strategy for evaluation of early and late ambulatory functions in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder that leads to mobility loss and life-threatening cardiac or respiratory complications. Quantitative ultrasound (QUS) envelope statistics imaging, which characterizes fat infiltration and fibrosis in muscles, has been extensively used for DMD evaluations. PURPOSE: Notably, changes in muscle microstructures also result in acoustic attenuation, potentially serving as another crucial imaging biomarker for DMD. Expanding upon the reference frequency method (RFM), this study contributes to the field by introducing the robust RFM (RRFM) as a novel approach for ultrasound attenuation imaging in DMD. METHODS: The RRFM algorithm was developed using an iterative reweighted least squares technique. We conducted standard phantom measurements with a clinical ultrasound system equipped with a linear array transducer to assess the improvement in attenuation estimation bias by RRFM. Additionally, 161 DMD patients, included in both a validation dataset (n = 130) and a testing dataset (n = 31), underwent ultrasound scanning of the gastrocnemius for RRFM-based attenuation imaging. The diagnostic performances for ambulatory functions and discrimination between early and late ambulatory stages were evaluated and compared with those of QUS envelope statistics imaging (involving Nakagami distribution, homodyned K distribution, and entropy values) using the area under the receiver operating characteristic curve (AUROC). RESULTS: The results indicated that the RRFM method more closely matched the actual attenuation properties of the phantom, reducing measurement bias by 50% compared to conventional RFM. The AUROCs for RRFM-based attenuation imaging, used to discriminate between early and late ambulatory stages, were 0.88 and 0.92 for the validation and testing datasets, respectively. These performances significantly surpassed those of QUS envelope statistics imaging (p < 0.05). CONCLUSIONS: Ultrasound attenuation imaging employing RRFM may serve as a sensitive tool for evaluating the progression of ambulatory function deterioration, offering substantial potential for the health management and follow-up care of DMD patients.

Second Order Dynamics Featuring Tikhonov Regularization and Time Scaling.

In a Hilbert setting we aim to study a second order in time differential equation, combining viscous and Hessian-driven damping, containing a time scaling parameter function and a Tikhonov regularization term. The dynamical system is related to the problem of minimization of a nonsmooth convex function. In the formulation of the problem as well as in our analysis we use the Moreau envelope of the objective function and its gradient and heavily rely on their properties. We show that there is a setting where the newly introduced system preserves and even improves the well-known fast convergence properties of the function and Moreau envelope along the trajectories and also of the gradient of Moreau envelope due to the presence of time scaling. Moreover, in a different setting we prove strong convergence of the trajectories to the element of minimal norm from the set of all minimizers of the objective. The manuscript concludes with various numerical results.

SARS-CoV-2 envelope protein alters calcium signaling via SERCA interactions.

The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.

The evolution of envelope function during coinfection with phylogenetically distinct human immunodeficiency virus.

BACKGROUND: Coinfection with two phylogenetically distinct Human Immunodeficiency Virus-1 (HIV-1) variants might provide an opportunity for rapid viral expansion and the emergence of fit variants that drive disease progression. However, autologous neutralising immune responses are known to drive Envelope (Env) diversity which can either enhance replicative capacity, have no effect, or reduce viral fitness. This study investigated whether in vivo outgrowth of coinfecting variants was linked to pseudovirus and infectious molecular clones' infectivity to determine whether diversification resulted in more fit virus with the potential to increase disease progression. RESULTS: For most participants, emergent recombinants displaced the co-transmitted variants and comprised the major population at 52 weeks postinfection with significantly higher entry efficiency than other co-circulating viruses. Our findings suggest that recombination within gp41 might have enhanced Env fusogenicity which contributed to the increase in pseudovirus entry efficiency. Finally, there was a significant correlation between pseudovirus entry efficiency and CD4 + T cell count, suggesting that the enhanced replicative capacity of recombinant variants could result in more virulent viruses. CONCLUSION: Coinfection provides variants with the opportunity to undergo rapid recombination that results in more infectious virus. This highlights the importance of monitoring the replicative fitness of emergent viruses.