Direct electron transfer (DET) between redox enzymes and electrodes is a crucial process in developing biosensors and cofactor-free bio electrosynthesis. However, due to unfavourable orientations, the absence of accessible redox centres, or long electron transfer distances, DET efficiency can be low. Here we present a systematic approach to better understand, evaluate and increase the DET capabilities of a formaldehyde dehydrogenase (F ald DH). F ald DH catalyses the reversible oxidation of formaldehyde to formate and is part of the CO 2 to methanol enzyme cascade. F ald DH from Burkholderia multivorans was fused to a DET capable domain, the soluble subunit of cytochrome b 562 from Escherichia coli. Fusion proteins with two different linker morphologies and various lengths were designed and biochemically and electrochemically characterised. The longest flexible linker had minor effects on biochemical constants and exhibited the highest increase in current density. We also identified an undesirable side-reaction between formaldehyde and basic amino acids to interfere with the electrochemical measurements and therefore normalised all currents to the percentage of basic amino acids on the solvent exposed surface area of the protein. This enabled us to present the first protein engineering approach to increase DET in this enzyme, resulting in a 1.6-fold increase in current density, compared to the wild-type enzyme. • Novel formaldehyde dehydrogenase fusion proteins were produced and characterised. • Fusion proteins are biochemically and electrochemically active. • Control experiments with non-enzymatic albumin also showed electroactivity. • The current density was normalised to the amount of basic amino acids. • This normalised data clearly indicates improvement in one fusion protein.

Development and validation of an indirect ELISA based on recombinant N-P fusion protein for detecting antibodies against parrot bornavirus in psittacine birds.

Recombinant fusion subunit vaccine provides enhanced immune protection against Cryptocaryon irritans infection in grouper.

Comparison of rod photoreceptor outer segment renewal in wild type and Tmem138-deficient mice using AAV-delivered Dendra2-tagged rhodopsin.

Immune efficacy evaluation of a PEDV spike protein-derived multi-epitope subunit vaccine candidate in mice.

Identification of three novel linear B-cell epitopes located on the glycoprotein of Micropterus salmoides rhabdovirus using monoclonal antibodies.

Membrane fusion between HIV and host cells requires interaction between the N-terminal and C-terminal repeat regions (NHR and CHR) of the gp41 envelope subunit. A deep hydrophobic pocket (HP) on the surface of NHR is considered crucial in this interaction. Targeting the viral gp41 CHR with stabilized trimeric NHR peptides or chimeric proteins effectively inhibits HIV infection. However, the contribution of each specific structural element, particularly the HP, in this mode of inhibition remains unclear. Here, we describe three chimeric proteins that structurally mimic the full NHR region interacting intramolecularly with CHR segments of varying lengths, either covering or exposing the HP. The intramolecular NHR-CHR interaction strongly stabilized all the chimeras. Binding analysis using a CHR-derived peptide and the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) combined with X-ray crystallography assessed the degree of exposure of the HP in the chimeras. Despite differences in HP accessibility, none of the chimeras displayed relevant inhibitory activity against several HIV-1 strains, suggesting that an exposed HP alone is insufficient to disrupt the NHR-CHR interaction in a viral context. The crystal structure of the ANS-chimera complex revealed the binding pose of ANS within the HP, while the overall CHR-NHR interaction closely resembled the canonical post-fusion six-helix-bundle structure. To our knowledge, this is the first crystallographic structure of a small molecule ligand independently bound to the HP. These findings provide insight into the role of the HP in NHR-based fusion inhibitors and guide the design of new, more focused and effective HIV inhibitors.

USP30 alleviates intestinal ischemia-reperfusion injury by deubiquitinating MFN2 mediating the mitochondrial endoplasmic reticulum pathway.

Glucagon-like peptide-1 (GLP-1), a 31-amino acid incretin hormone, is widely used in the treatment of type 2 diabetes mellitus due to its glucose-dependent insulinotropic activity. However, its small size makes it highly prone to proteolytic degradation in microbial expression systems such as Escherichia coli, leading to reduced manufacturing yield. While fusion to cleavable protein tags can improve peptide stability during purification, excessively large tags often compromise the overall yield, especially when the target peptide is smaller than the fusion partner. To overcome this limitation, we have engineered 11 cleavable fusion tag constructs (LP1-LP11) for recombinant expression of Arg34-GLP-1(7-37) (liraglutide precursor) in E. coli. The 11 constructs differed only in the tags. The expression vector contained a T7 leader sequence, affinity tags (6×His/6×Arg), inclusion body tags (11-125 amino acids), and TEV protease cleavage sites. Among the 11 tags, LP8 with a compact 4.0 kDa tag achieved the highest expression, yielding 133 mg/L of fusion protein and a calculated liraglutide precursor yield of 60 mg/L based on mass fraction (45% of fusion mass), with an actual recovered yield of 14.6 mg/L after RP-HPLC purification, largely due to efficient inclusion body formation (>95% insolubility) and enhanced translational initiation driven by the T7 leader sequence. The purified peptide's identity and sequence integrity were confirmed by LC/MS analysis. The primary advantage of this approach is mass fraction optimization which focuses on minimizing fusion-tag mass to maximize yield relative to the tag size without compromising inclusion-body formation thereby providing a scalable and economical approach for GLP-1 analogs and potentially other peptide-based biopharmaceuticals.

Development of a NanoBiT based high throughput screening assay for discovery of NOS1-NOS1AP interaction inhibitors.

Mitofusin 2 in central nervous system disorders: Roles in mitochondrial dynamics and therapeutic Implications.

Mitochondrial fusion orchestrated by hepatitis E virus couples pro-viral autophagy and innate immune evasion for efficient replication.

Design, synthesis and pharmacological evaluation of phenylthiophene-2-carboxamide-based RSV fusion protein inhibitors.

Chlamydomonas reinhardtii, which is a unicellular photosynthetic eukaryote, has long served as a model microalga for fundamental biological research and biotechnological applications. Recently, it has attracted attention as a promising biological resource for the sustainable production of bio-oils and high-value biomolecules. To enhance the biotechnological utility of this species, various genetic engineering tools have been developed in recent years. In this study, the tetracycline repressor (TetR)-based transactivation system, which is widely used in mammalian and other eukaryotic cells, was repurposed to establish a synthetic transcriptional activation system for exogenous gene expression in Chlamydomonas. We first constructed a transient expression-based evaluation platform to screen for transcriptional activation domains (TADs) that are functional in Chlamydomonas. Among the analyzed TADs, VP192 (tandem repeat of 12 copies of the core VP16 domain) had the highest transcriptional activity when fused to either TetR or Gal4 DNA-binding domains. Furthermore, fusion proteins comprising VP192 and either TetR or reverse TetR enabled doxycycline-dependent regulation of transgene expression in a dose-dependent manner. Notably, transcriptional inducibility was maintained even when the tetracycline-responsive element (TRE) was fused to the HSP70A promoter. Combining TetR-VP192 and this synthetic chimeric promoter (TRE-PHSP) yielded transgene expression levels that exceeded those resulting from the strong HSP70A/RbcS2 hybrid promoter by more than 14-fold. These findings suggest that artificial transcription factors and engineered promoters provide a versatile molecular toolkit for regulating exogenous gene expression in Chlamydomonas.

Design, synthesis, and activity evaluation of RET protein degradation based on PROTAC and HyTTD techniques.

Next-generation interleukin-enhanced immunotherapies: From molecular engineering to armored T-cell architectures.

Impact of injection time and protein modality on particle formation when using closed system transfer devices.

Heterosis, characterized by enhanced resistance and yield, has been widely utilized in watermelon breeding. However, our understanding of the regulatory mechanisms underlying male-sterile phenotypes in watermelon remains limited. Here, we determined that the miR159a targets ClMYB33 to regulate anther dehiscence, leading to male sterility in watermelon. Both overexpression of Cl-miR159a (OE-miR159a) and knockout of ClMYB33 (clmyb33) in watermelon suppressed the degradation of septum and stomium tissues, thereby impairing anther dehiscence and preventing successful pollen release. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, ClPG1 and ClQRT2 were identified as downstream target genes of ClMYB33; both were positively regulated by ClMYB33. Both ClPG1 and ClQRT2 exhibited polygalacturonase (PG) activity in vivo. The knockout of ClQRT2 led to reduced PG activity and a failure in anther dehiscence. Furthermore, the GST-ClQRT2 fusion protein was capable of rescuing the indehiscent anther phenotype observed in both OE-miR159a and clmyb33 plants. Our results reveal a new mechanism by which the miR159a-ClMYB33 module regulates anther dehiscence bymediating PG activity, and provide a new molecular tool to create male sterility in watermelon.

Newcastle disease virus (NDV) infects a wide range of hosts, including poultry and wild birds. However, most research has focused on poultry-derived strains, with limited studies on the biological characteristics of NDV isolated from wild birds. This study investigated genotype IX NDV strains from wild birds. Genetic evolution and viral ecology analyses suggest possible poultry-to-wild-bird transmission of this genotype. Most of the genotype IX NDV strains, isolated from asymptomatic wild birds, were highly pathogenic in chickens, but one strain isolated from spotted dove exhibited attenuated virulence. This dove-derived strain showed the highest homology with a virulent strain from Eurasian blackbird, differing by only 12 amino acids across proteins. Using reverse genetics, we identified the viral HN protein as a critical determinant of virulence in the dove strain. The coexistence of F110 and G116 residues in the HN protein was associated with reduced HN cell-surface abundance and downstream decreases in viral membrane fusion activity, replication capacity, and tissue tropism. Computational modeling further suggests that the F110/G116 combination may be linked to a local α-helix within the HN head-stalk linker region, providing a plausible structural context for these effects. Crucially, this dove strain's fusion protein retained the furin-cleavable, virulent-type "¹¹²RRQRRF¹¹⁷" cleavage site. Immunizing chickens with this strain induced high antibody titers within 1 week, and titers persisted at high levels for 13 weeks and provided complete protection against virulent challenge at both early and late post-immunization time points. Our findings uncover an HN-linked attenuation mechanism centered on residues 110 and 116, primarily through their effects on HN cell-surface abundance and downstream fusion activity, offering new insights into the development of immunogenic NDV vaccines with reduced virulence.IMPORTANCEWild birds, as natural reservoirs of Newcastle disease virus (NDV), harbor a diverse range of viral strains. However, research on these strains remains limited. Here, we show that genotype IX NDV detected in wild birds most likely reflects possible poultry-to-wild bird transmission rather than long-term endemic circulation in wild populations. Using isolates derived from wild birds, we report a novel attenuation mechanism involving structural modification of the HN protein's head-neck linker region. Therefore, research on wild bird-derived isolates not only provides insights into the epidemiological dynamics of NDV transmission but also facilitates the identification of novel attenuated strains, which could aid in the development of high-efficacy NDV vaccines and NDV-based viral vectors.

Glioblastoma is a lethal brain tumor that is resistant to conventional therapies. Here we present a non-lytic replicating retrovirus (RRV) that delivers an IL-15-receptor-linked fusion protein (RLI) superagonist directly into glioblastoma cells, creating localized immunotherapy biofactories. In orthotopic mouse models, RRV-RLI dramatically suppresses tumor growth, prolongs survival, and induces lasting remission with immunologic memory. Mechanistically, we observe increased CD8⁺ T cell and natural killer cell infiltration and activation, alongside elevated antigen presentation pathways. Combining RRV-RLI with temozolomide, which is standard-of-care chemotherapy for glioblastoma, enhances antitumor immunity. T cell receptor sequencing reveals a polyclonal repertoire of T cells, enhanced by combining RRV-RLI with temozolomide. Analysis of the T-cell repertoire suggests it to be directed against tumor rather than viral antigens, supporting the specificity and re-applicability of our approach. These findings illustrate that RRV-RLI reprograms glioblastoma into an immunostimulatory hub, offering a viral immunotherapy against glioblastoma and potentially other therapy-resistant solid tumors.