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.

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.

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.

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.

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).

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.

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.

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.

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.

Regulatory role of the Cpx ESR in bacterial behaviours.

The envelope demarcates the boundary between bacterial cell and its environment, providing a place for bacteria to transport nutrients and excrete metabolic waste, while buffering external environmental stress. Envelope stress responses (ESRs) are important tools for bacteria to sense and repair envelope damage. In this review, we discussed evidence that indicates the important role of the Cpx ESR in pathogen-host interactions, including environmental stress sensing and responses, modulation of bacterial virulence, antimicrobial resistance, and inter-kingdom signaling.

3,3'-Diindolylmethane disrupts the endoplasmic reticulum and nuclear envelope in Schizosaccharomyces pombe.

3,3'-Diindolylmethane is recognized for its anti-cancer activities in various pathways, though its mechanism remains to be fully elucidated. Previous studies have shown that 3,3'-Diindolylmethane disturbed the localization of Cut11, a nuclear pore complex subunit in Schizosaccharomyces pombe. This study further reveals that in Schizosaccharomyces pombe, 3,3'-Diindolylmethane also disrupts other components of nuclear envelope, causing GFP-NLS leakage, making it evident that 3,3'-Diindolylmethane disrupts the nuclear envelope. 3,3'-Diindolylmethane also disturbs the localization of GFP-ADEL and Ost4, which are endoplasmic reticulum lumen proteins and membrane proteins respectively, suggesting the function of 3,3'-Diindolylmethane on endoplasmic reticulum disturbance. The nuclear envelope repairment, normal nuclear envelope physical properties, and lipid metabolism homeostasis are crucial for cell survival in the presence of 3,3'-Diindolylmethane. These findings provide new insights into the understanding and development of 3,3'-Diindolylmethane as an anti-cancer agent.

The Furin Protease Dependence and Antiviral GBP2 Sensitivity of Murine Leukemia Virus Infection Are Determined by the Amino Acid Sequence at the Envelope Glycoprotein Cleavage Site.

Host restriction factor GBP2 suppresses the replication of the ecotropic Moloney murine leukemia virus (E-MLV) by inhibiting furin protease, which cleaves the viral envelope glycoprotein (Env) into surface (SU) and transmembrane (TM) subunits. We analyzed the impacts of GBP2 on the infection efficiency mediated by MLV Envs of different strains of ecotropic Moloney, polytropic Friend, amphotropic, and xenotropic MLV-related (XMRV) viruses. Interestingly, the Envs of ecotropic Moloney and polytropic Friend MLV were sensitive to the antiviral activity of GBP2, while XMRV and amphotropic Envs showed resistance. Consistent with the sensitivity to GBP2, the amino acid sequences of the sensitive Envs at the SU-TM cleavage site were similar, as were the sequences of the resistant Envs. SU-TM cleavage of the GBP2-sensitive Env protein was inhibited by furin silencing, whereas that of GBP2-resistant Env was not. The substitution of the ecotropic Moloney cleavage site sequence with that of XMRV conferred resistance to both GBP2 and furin silencing. Reciprocally, the substitution of the XMRV cleavage site sequence with that of the ecotropic sequence conferred sensitivity to GBP2 and furin silencing. According to the SU-TM cleavage site sequence, there were sensitive and resistant variants among ecotropic, polytropic, and xenotropic MLVs. This study found that the dependence of MLV Env proteins on furin cleavage and GBP2-mediated restriction is determined by the amino acid sequences at the SU-TM cleavage site.

Treatment of distal third tibial fractures with anterior soft tissue compromise through the posterolateral approach: Distal Third Tibial Fractures via the Posterolateral Approach.

Distal third tibial fractures associated with anterior soft tissue compromise are a predictor of more complications and poor prognosis. The study aimed to introduce the treatment of such fractures through the posterolateral approach. From March 2020 and January 2022, 32 patients with distal third tibial fractures were plated through the posterolateral approach due to concurrent closed anterior soft tissue compromise. There were 30 male and 2 female patients with the mean age of 33 years (range, 20-53 years). The reduction quality of diaphyseal fractures was good (n=30) and acceptable (n=2). The reduction quality of articular fragments was anatomic (n=21), good (n=6), and fair (n=1). All anterior soft tissue injuries healed without surgical intervention. Follow-ups lasted 28 months (range, 25-34 months). The mean dorsiflexion of the injured and uninjured ankles were 17.8°±5.4° and 24.5°±6.6°, respectively (P<0.05). The mean plantar flexion of the ankles were 42°±8.8° and 46°±12.9°, respectively (P>0.05). The mean inversion of the injured and uninjured ankles were 15°±13.3° and 19°±12.4°, respectively (P<0.05). The mean eversion of the injured and uninjured ankles were 27.8°±16.9° and 32.9°±14.3°, respectively (P>0.05). The mean American Orthopaedic Foot and Ankle score was 90 (range, 68-100). Distal third tibial fractures with anterior soft tissue compromise can be plated through the posterolateral approach, resulting in good functional outcomes and minimum complications. LEVEL OF EVIDENCE: Therapeutic study, Level IV.

Management of Thick Skin in Rhinoplasty.

Rhinoplasty in thick skin patients is challenging because the skin soft tissue envelope (S-STE) is more inelastic, and has a tendency for prolonged postsurgical edema, increased dead space formation, and underlying scar tissue formation. Changes in the S-STE will have an impact on how the final rhinoplasty result will look. When performing surgery, approaches should be targeted to the underlying bony-cartilaginous framework and the S-STE to obtain consistent, improved long term results. In this article, 3 experts will be discussing up to date medical, topical, and surgical management key points, as well as diagnostic options and post-operative treatments.

Immunogenicity and Protective Efficacy of Baculovirus-Expressed SARS-CoV-2 Envelope Protein in Mice as a Universal Vaccine Candidate.

The envelope (env) protein of SARS-CoV-2, a pivotal component of the viral architecture, plays a multifaceted role in viral assembly, replication, pathogenesis, and ion channel activity. These features make it a significant target for understanding virus-host interactions and developing vaccines to combat COVID-19. Recent structural studies provide valuable insights into the conformational dynamics and membrane topology of the SARS-CoV-2 env protein, shedding light on its functional mechanisms. The strong homology and highly conserved structure of the SARS-CoV-2 env protein shape its immunogenicity and functional characteristics. This study examines the ability of the recombinant SARS-CoV-2 env protein to stimulate an immune response. In this study, recombinant envelope proteins were produced using the baculovirus expression system, and their potential efficacy was evaluated in both in vivo and in vitro models. Our results reveal that the env protein of SARS-CoV-2 stimulates humoral and cellular responses and highlight its potential as a promising vaccine candidate for combating the ongoing pandemic.

A Novel Bi-Directional Channel for Nutrient Uptake across Mycobacterial Outer Envelope.

Nutrients are absorbed by special transport proteins on the cell membrane; however, there is less information regarding transporters across the mycobacterial outer envelope, which comprises dense and intricate structures. In this study, we focus on the model organism Mycolicibacterium smegmatis, which has a cell envelope similar to that of Mycobacterium tuberculosis, as well as on the TiME protein secretion tube across the mycobacterial outer envelope. We present transcriptome results and analyze the protein compositions of a mycobacterial surface envelope, determining that more transporters and porins are induced to complement the deletion of the time gene in Mycolicibacterium smegmatis. The TiME protein is essential for nutrient utilization, as demonstrated in the uptake experiments and growth on various monosaccharides or with amino acids as the sole carbon source. Its deletion caused bacteria to be more sensitive to anti-TB drugs and to show a growth defect at an acid pH level, indicating that TiME promotes the survival of M. smegmatis in antibiotic-containing and acidic environments. These results suggest that TiME tubes facilitate bi-directional processes for both protein secretion and nutrient uptake across the mycobacterial outer envelope.

The Discovery of Membrane Vesicle Biogenesis in the Polyhydroxybutyrate-non-producing Mutant Strain of Cupriavidus necator H16.

Extracellular membrane vesicles (MVs) caused by the artificial production of polyhydroxybutyrate (PHB) were previously detected in Escherichia coli. We herein observed MV biogenesis in the mutant strain (-PHB) of the natural PHB producer, Cupriavidus necator H16. This inverse relationship was revealed through comparative electron microscopic ana-lyses of wild-type and mutant strains. Based on these results, we speculate that a physiological relationship exists between MV biogenesis and PHB biosynthesis. Therefore, we propose the potential of MV biogenesis as a fermentative "stress marker" for monitoring the performance of target polymer-producing microbial platforms.