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The dendritic cell-targeting peptide (DCP) trimer sequence has previously shown to enhance specific IgG induction. This study aims to investigate the expression of rabies virus glycoprotein (RVG) in eukaryotic cells through cloning in the pCDNA3.1 + vector, alongside the fusion of a DCP. The DCP trimer sequence was fused to the C-terminal of the RVG to improve the immune response. The construct was designed to include a His-tag for protein purification and an enterokinase (EK) cleavage site for separation of the tag from the recombinant protein. The RVG gene was amplified from the rabies virus genome via RT-PCR, and cloning was performed using BamHI and EcoRI restriction enzymes. The recombinant plasmid (RVG- pCDNA3.1+) was transfected into BHK-21 cells using lipofection, and the expression of the recombinant protein was confirmed through SDS-PAGE and western blotting using anti-His antibodies. The His-tagged recombinant protein was purified using Ni-NTA resin. Immunogenicity of the recombinant protein was investigated through mouse inoculation and analysis of the serum samples using RFFIT and ELISA methods. The results indicated successful cloning and expression of the RVG-DCP fusion protein in eukaryotic cells, with potential applications in rabies vaccine development. The results suggests a novel method for enhancing the immunogenicity of viral glycoproteins through the use of dendritic cell-targeting peptides.

Few studies have reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein may suppress cancer cell growth. Here, we investigated the effect of the SARS-CoV-2 spike protein in A549 lung cancer cells using recombinant spike protein (SP), spike protein transfection (SPT), and pseudo-SARS-CoV-2 virus (PSV). To evaluate its anticancer effects and associated molecular changes, we performed RNA sequencing, colony formation, immunofluorescence, cell viability, migration, FACS, western blotting, 3D spheroid, molecular docking, siRNA knockdown, qRT-PCR, apoptosis assays, and computational interaction analyses. Spike protein significantly inhibited the long-term growth of A549 cells. Among the delivery methods, PSV showed the most potent anticancer effect, followed by SPT and SP, as evidenced by reduced migration, spheroid growth, and increased sub-G1 arrest. RNA-Seq identified two differentially expressed lncRNAs, with MEG3 upregulated and BCYRN1 downregulated following spike-related treatment. Functional experiments showed that BCYRN1 knockdown or MEG3 overexpression reproduced key spike protein-induced antiproliferative and pro-apoptotic effects, including increased cleaved caspase-3 expression. Computational analyses suggested possible interactions between the Spike protein and these lncRNAs. In addition, actinomycin D (ActD) chase experiments indicated altered transcript dynamics of MEG3 and BCYRN1 under spike-related conditions. Collectively, these findings suggest that SARS-CoV-2 spike protein exerts antitumor activity in A549 cells and may contribute to the regulation of MEG3 and BCYRN1. Further studies, including formal rescue and biochemical interaction assay, will be required to establish causality and direct molecular mechanisms.

Interactomics aims at identifying and characterizing in a comprehensive way the complete set of protein-protein interactions that occur within a cell, tissue, or organism. In this chapter, we cover the most relevant experimental and computational approaches that are used in the field to assess the interactome of a specific protein or of all the proteins in a defined compartment. We address their applicability as well as their advantages and disadvantages. We then describe the different databases available to researchers interested in dissecting protein-protein interactions and finally briefly summarize the several attempts that have been made to identify the human interactome, i.e., the complete set of protein interactions that occur within a human cell.

Protein secondary structure prediction represents an important intermediate step between a protein's linear amino acid sequence and its three-dimensional structure, with broad implications for synthetic biology, drug development, and disease research. Although experimental techniques such as X-ray crystallography provide highly accurate structural information, they are labor-intensive, time-consuming, and costly, which has motivated the development of computational alternatives. Early machine-learning approaches to this problem were limited in their ability to capture complex sequence-structure relationships. The introduction of convolutional and recurrent neural networks improved hierarchical feature extraction, and predictive performance advanced further with transformer-based architectures such as AlphaFold2. This review outlines recent advances in hybrid model design, benchmark datasets, and evaluation metrics for protein secondary structure prediction. We also discuss current methodological limitations, including data dependency and dataset bias, and outline future directions such as cross-species validation, uncertainty-aware modeling, and the still-emerging potential of incorporating heterogeneous biological data into next-generation PSSP frameworks.

Aptamers, as single-stranded DNA (ssDNA) or RNA oligonucleotides, are pivotal in biosensing due to their high affinity. However, excessive lengths of these nucleic acid probes can impair their binding affinity and target recognition efficiency. Traditional optimization methods, such as static structural modeling, fail to capture the dynamic interactions between aptamers and biological macromolecules. Therefore, optimizing aptamer length to enhance affinity while maintaining effective target recognition is crucial. Here, we employed 600ns molecular dynamics (MD) simulations using the amberff14sb and parmbsc1 force fields, alongside molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculations to optimize the binding affinity of a ssDNA aptamer targeting the hemagglutinin-neuraminidase (HN) protein-a critical surface receptor of Newcastle disease virus (NDV) responsible for viral attachment and entry. By systematically truncating the aptamer sequence guided by normalized criteria to eliminate length bias, we identified a 10-nucleotide variant (fqh-2) that exhibited a hydrogen bond efficiency ratio (HBER) of 1.055 and a binding free energy efficiency ratio (BFEER) of -5.124 kcal/mol, reflecting enhanced interactions with the HN protein. Furthermore, a graphene oxide (GO)-based fluorescence quenching assay confirmed a threefold increase in binding affinity for the optimized aptamer, aligning with computational predictions. This study not only provides a dynamic structure-guided framework for aptamer optimization but also lays a theoretical foundation for further advancements in optimizing and tailoring aptamers for specific applications.

Limitations in genome editing of avian species, including chicken, due to the inaccessibility of one-cell zygotes, have led to the manipulation of primordial germ cells (PGCs) as the primary approach for generating genetically engineered birds. Although plasmid-mediated delivery of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used, it has several limitations, including delayed nuclease activation, increased off-target effects, cytotoxicity, and the inability to apply in vivo selection strategies. Collectively, these limitations highlight the need to develop strategies that achieve high on-target activity at the initial editing step. In this study, we compared plasmid- and ribonucleoprotein (RNP)-mediated CRISPR/Cas9 delivery in Leghorn male hepatoma (LMH) cells and in PGCs targeting three loci: deleted in azoospermia like (DAZL), chicken vasa homologue (CVH), and stimulated by retinoic acid 8 (STRA8). RNP delivery showed comparable or higher cell viability and editing efficiency than plasmid delivery in both cell types. Insertion/deletion (indel) profiling revealed broader and more diverse mutation patterns with RNPs, consistent with a shift in repair pathway engagement toward microhomology-mediated end joining (MMEJ), which favors larger deletions. Off-target analysis further showed substantially reduced off-target editing and high specificity with RNP delivery. Together, these findings demonstrate that RNP delivery improves efficiency, reduces cytotoxicity, and improves precision, providing a more reliable platform for chicken genetic engineering.

In the absence of a definitive gold standard for diagnosing latent tuberculosis infection (LTBI) in healthcare workers (HCWs), alternative specific biomarkers are needed. This longitudinal cohort study with baseline testing evaluated the diagnostic performance of stimulated plasma interferon-&#x3b3; inducible protein 10 (IP-10) for LTBI detection among HCWs in a high tuberculosis (TB)-burden setting, using interferon-&#x3b3; release assays (IGRAs) as the reference standard. The study was conducted at Songklanagarind Hospital, Thailand, between 2021 and 2024, and included 64 healthy HCWs. Serum and stimulated plasma samples were collected, and IP-10 and IFN-&#x3b3; concentrations were measured by ELISA. Nine HCWs (14.0%) were LTBI-positive. Stimulated plasma IP-10, but not serum IP-10, showed significant correlations with IFN-&#x3b3; levels in TB1 (r&#x2009;=&#x2009;0.719) and TB2 (r&#x2009;=&#x2009;0.768; p&#x2009;<&#x2009;0.0001) tubes. At cut-off values of &#x2265;&#x2009;2,980.0 pg/mL for TB1 and &#x2265;&#x2009;3,108.0 pg/mL for TB2, sensitivity was 100%, specificity was 96.0% and 94.5%, and overall accuracy was 96.8% and 95.3%, respectively. Based on either IGRA or stimulated plasma IP-10 cut-off levels, five of nine LTBI cases declined treatment; among these, one individual (20.0%) progressed to active pulmonary TB 16 months later. Stimulated plasma IP-10 shows high diagnostic performance for LTBI detection among HCWs and may serve as an alternative for LTBI screening in high-TB-incidence with universal BCG vaccination.

Exercise provides significant benefits for most women, but adverse effects on reproduction can and do occur. Increased frequency, duration, and intensity of exercise are associated with a higher risk of female infertility. In particular, exercise-induced reproductive dysfunction is linked to alterations in the hypothalamic-pituitary-gonadal (HPG) axis. However, the role of meteorin-like protein (metrnl), as an exercise-induced myokine, in female reproductive function and the HPG axis is not yet fully understood. In this study, we investigated the effects of metrnl, compared with a swimming exercise protocol, on sexual motivation and the HPG axis in female rats. Female rats with regular estrous cycles were randomly allocated into three groups (n&#x2009;=&#x2009;9 per group), and subjected to deionized water (1 mL/kg), metrnl (1&#xa0;&#xb5;g/day) or swimming-based moderate-intensity exercise (30&#xa0;min/day, 5 days/week) for about 19 consecutive days. The results showed that metrnl administration reduced the time spent near the male rat and the male preference ratio, indicators of sexual motivation. It also decreased hypothalamic GnRH expression (in the median eminence and preoptic area), serum concentrations of reproductive hormones (follicle-stimulating hormone and anti-M&#xfc;llerian hormone), and the numbers of ovarian follicles (primordial and primary follicles), similar to the effects observed after the swimming exercise protocol. Furthermore, metrnl concentrations in serum remained unchanged in female rats treated with metrnl and exercise. In conclusion, metrnl treatment reduced sexual incentive motivation and altered HPG axis regulation in female rats. These findings suggest that exercise-induced metrnl may be a potential exerkine associated with changes in female sexual motivation and HPG axis regulation due to exercise.

Extreme high temperatures significantly threaten insect development and reproduction. Small heat shock proteins (sHSPs) are crucial for thermal adaptation, but their specific roles in reproduction and heat stress response remain incompletely understood. This study investigates SfHSP19.8, a testis-enriched sHSP in the fall armyworm (Spodoptera frugiperda), to elucidate its function. Testis-specific expression of SfHSP19.8 occurred during the 6th- instar larval and pupal stages. Homozygous knockout mutants were generated using CRISPR/Cas9 technology. Transcriptomic analysis of 6th-instar larval testes revealed dysregulation of genes in steroid hormone biosynthesis and detoxification pathways. However, knockout did not impair adult male fertility, testis morphology or sperm count under standard conditions. SfHSP19.8 expression was strongly induced by heat shock across developmental stages. Mutants exhibited severely compromised thermotolerance under heat stress, including complete egg hatching failure, high larval mortality, prolonged larval development, and reduced adult longevity and reproductive fitness. These findings demonstrate that SfHSP19.8 is essential for systemic thermotolerance across life stages and maintains reproductive function specifically under thermal challenge, while being dispensable for basal male reproduction under optimal conditions. SfHSP19.8 represents a potential target for pest control strategies in the context of future climate warming. &#xa9; 2026 Society of Chemical Industry.

Heart failure (HF) is a major complication of metabolic dysfunction-associated steatotic liver disease (MASLD). The C-reactive protein-triglyceride glucose index (CTI), which reflects systemic inflammation and insulin resistance, is linked to cardiovascular outcomes, but its predictive value for HF in MASLD patients remains unclear. We aimed to evaluate the baseline and cumulative CTI (CumCTI) in relation to incident HF and explore blood pressure as a potential mediator. We included 26,499 participants with MASLD from the Kailuan Study. The baseline and CumCTI values were calculated and examined for associations with HF via Cox regression. Restricted cubic splines were used to assess dose&#x2012;response patterns. Predictive performance was evaluated using C-index, reclassification metrics, time-dependent ROC curves, and calibration analyses. During a median follow-up of 16.0 years, 1,158 HF events occurred. A higher CTI was independently associated with increased HF risk (HR for Q4 vs. Q1: 1.78; 95% CI: 1.48-2.11; P&#x2009;<&#x2009;0.001), with each 1-SD increase corresponding to a 27% greater risk (HR 1.27; 95% CI: 1.19-1.35). CumCTI showed stronger associations with HF and modestly improved model discrimination and reclassification compared with conventional risk factors. Time-dependent ROC analyses showed slight increases in AUC at 5 and 10 years after adding CumCTI (0.782 to 0.783 and 0.763 to 0.767, respectively), with good calibration between predicted and observed risks. Mediation analysis indicated that systolic and diastolic blood pressure explained only a small proportion of the CTI-HF association. Associations were stronger for CumCTI in participants aged&#x2009;<&#x2009;60 years. These findings suggest that a higher CTI, especially cumulative exposure, is independently associated with HF risk in MASLD and may provide complementary information for cardiovascular risk assessment.

To assess the combination of high dose interleukin 2 (HD IL2) and nivolumab in patients with checkpoint inhibitor-refractory melanoma or renal cell carcinoma (RCC), we performed a single-arm, Simon two-stage phase II trial for patients with unresectable stage III or IV melanoma or any histology RCC. The primary endpoint was overall response rate (ORR); secondary endpoints were adverse events and progression-free survival (PFS), defined as time to radiographic or clinical progression, whichever occurred first. Five patients enrolled, three with melanoma and two with RCC. Median age at registration was 47 (35-77) years. Two patients (40%) had treated or asymptomatic brain metastases, and one (20%) had liver metastases. Median prior lines of systemic therapy per patient were 3 (2-5). Patients completed a median of 1 (0.5-3) treatment cycle, involving two admissions for HD IL2 administration and two doses of nivolumab. ORR was 20%. Median PFS was 1.4 (0.8-45.0) months. Time to next therapy for each living patient was 2.5 and 45.6 months for local modalities, and 2.0, 2.3, and 45.9 months for systemic therapies. Adverse events reflected the known toxicity of HD IL2. One treatment-related death occurred, leading to trial termination. The combination of HD IL2 and nivolumab resulted in a durable response in one of five patients with PD-1-refractory advanced melanoma or RCC. Use of HD IL2 remains limited by its toxicity; augmented IL2 formulations and administration strategies are needed. ClinicalTrials.gov ID: NCT03889782 (Registered 6/16/2019; https://clinicaltrials.gov/study/NCT03889782).

Multispecificity involves high-affinity binding to multiple related ligands while preserving discrimination against other ligands. The binding of tick evasins to human CC chemokines exemplifies key principles of multispecific binding: a rigid core recognising conserved features of all targets, with flexible peripheral regions enabling discrimination within the target family.