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Naja kaouthia , or the monocled cobra, is one of the most medically important snakes in Thailand, responsible for approximately 17% of snakebite cases. Conventional horse-derived antivenoms are lifesaving, yet they may trigger severe allergic reactions and exhibit batch to batch variability. Nanobodies (VHH) are promising alternatives as recombinant antivenoms having demonstrated the ability to neutralize snake venom both in vitro and in vivo . However, a major challenge in developing them is the diverse and complex composition of snake venoms, which requires therapies capable of targeting multiple toxins. To address this, we developed a bispecific VHH that simultaneously targets the two main toxins in N. kaouthia venoms, α-neurotoxin (αNTX) and phospholipase A 2 (PLA 2 ), fused to a human IgG Fc domain (bispecific VHH-Fc), which was selected to prolong serum half-life and reduce the immunogenicity risks associated with animal-derived antivenoms . The bispecific VHH-Fc, along with two monospecific nanobodies (VHH-αNTX-Fc and VHH-PLA 2 -Fc), was expressed in Chinese hamster ovary (CHO) cells and purified from culture supernatant after 5–6 days. Immunoblotting confirmed the successful expression and Fc fusion of these constructs, as detected by anti-human IgG-Fc antibodies conjugated to horseradish peroxidase (HRP). Importantly, antigen-binding assays demonstrated that the bispecific VHH-Fc exhibited the the strongest binding signal to crude N. kaouthia venom compared to the monospecific nanobodies. In in vivo murine neutralization assays, the bispecific VHH-Fc showing higher survival than equine-derived antivenom (33%) and comparable efficacy to a VHH-Fc cocktail under the tested conditions. Complete protection was achieved at higher doses. These results demonstrate that the bispecific VHH-Fc can be efficiently produced in a mammalian expression system and possesses strong binding and neutralizing activity against N. kaouthia venom under the defined experimental conditions. Our findings support the bispecific VHH-Fc as a promising next-generation therapeutic candidate for the treatment of snakebite envenoming, while highlighting the importance of integrating binding and functional assays when evaluating antibody efficacy.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG 1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A ® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG 1 /IgG 2a ) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A ® , eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A ® , respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5–15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10–15 µM) for 12 h. Moreover, 5–15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin β1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Background Drug resistance remains a major obstacle to effective lung cancer treatment. Cisplatin, which is a platinum-based chemotherapeutic agent, is widely used as a first-line treatment for non-small cell lung cancer (NSCLC); however, its clinical efficacy is often limited by the development of resistance. MicroRNAs (miRNAs) are key regulators of the epigenetic landscape and have been reported to play critical roles in various cancer-related processes. This study aimed to identify potential miRNAs associated with the DNA damage response (DDR), which is a major mechanism underlying cisplatin sensitivity, and to investigate the role of miRNAs in modulating the cisplatin response. Methods Differential expression analysis of DNA damage response-related miRNAs was performed using lung cancer datasets from The Cancer Genome Atlas (TCGA). The role of miR-145-5p in cisplatin responsiveness was evaluated by transfecting lung cancer cell lines with a miR-145-5p mimic. The IC₅₀ of cisplatin was assessed using the MTT assay. Cisplatin-induced apoptosis was investigated through apoptosis assays. In addition, changes in apoptosis markers and associated signaling pathways were analyzed by immunoblotting. The potential target of miR-145-5p was identified by integrating data from multiple miRNA databases. The regulatory relationship between miR-145-5p and its target in relation to cisplatin sensitivity was further validated using luciferase reporter assays, RNA interference, and rescue experiments. Results miR-145-5p was identified as a strong candidate, as it was observed to significantly downregulated in lung tumor tissues and associated with poor prognosis. In vitro experiments demonstrated that miR-145-5p expression was positively correlated with cisplatin sensitivity across a panel of NSCLC cell lines and that its overexpression reduced the cisplatin IC 50 while increasing apoptosis. By integrating target prediction and validation by a luciferase reporter assay, CAMSAP2 was identified as a potential direct target of miR-145-5p. CAMSAP2 expression was negatively associated with cisplatin responsiveness, and rescue experiments confirmed that miR-145-5p could enhance cisplatin-induced apoptosis by downregulating CAMSAP2. Conclusions miR-145-5p directly targets and suppresses CAMSAP2 , sensitizing lung cancer cells to cisplatin. These findings highlight the miR-145-5p/CAMSAP2 regulatory axis as a critical modulator of cisplatin sensitivity and a potential therapeutic target for overcoming drug resistance in lung cancer.

Background/Aim: Box A is a highly conserved DNA-binding domain of high-mobility group box 1 (HMGB1) and has been shown to reverse senescence and aging features in many cell models. We investigated whether the activation of box A can influence stem cell properties. Materials and Methods: Human dermal papilla (DP) cells and primary human white pre-adipocytes (HWPc) were employed as mesenchymal cell models. Box A-overexpressing plasmids were used to induce cellular box A expression. mRNA and protein levels of stemness markers POU class 5 homeobox 1 pseudogene 5 (OCT4, HGNC: 9221), Nanog homeobox (NANOG, HGNC: 20857), and SRY-box transcription factor 2 (SOX2, HGNC:11195) in DP cells and HWPc were measured by real-time polymerase chain reaction and immunofluorescence analysis, respectively. Results: Transfection efficiency of box A-overexpressing plasmid was 80% and 50% in DP cells and HWPc, respectively. The proliferative rate of both cell types significantly increased 72 h after transfection. Levels of OCT4, NANOG and SOX2 mRNA and protein expression were significantly increased in box A-transfected DP cells and HWPc compared to empty plasmid-transfected cells. Immunofluorescence analysis confirmed the induction of OCT4, NANOG and SOX2 protein expression in response to box A in DP cells and HWPc. OCT4 and SOX2 were expressed in both the nuclear and cytoplasmic compartments, while NANOG was intensely located in the nucleus of box A-transfected cells. Conclusion: Our findings suggest that box A may potentially enhance stemness, which may have significant benefits in improving stem cell function due to aging processes and disease. This research may have implications for regenerative medicine applications.

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion.

Particular matter 2.5 (PM2.5) pollution is associated with senescence induction. Since the impact of PM2.5 on stem cell senescence and potential compounds capable of reversing this process are largely unknown, this study aimed to examine the senescence effects of PM2.5 on dermal papilla (DP) stem cells. Additionally, we explored the reversal of these effects using natural product-derived substances, such as resveratrol (Res) or Emblica fruits, soybean, and Thunbergia Laurifolia (EST) extract. Cell senescence was determined using the β-Galactosidase (SA-β-gal) assay. The senescence-associated secretory phenotype (SASP) was detected using real-time RT-PCR. For senescence markers, the mRNA and protein levels of p21 and p16 were measured using real-time RT-PCR and immunofluorescence analysis. Subtoxic concentration of PM2.5 (50 μg/ml) induced senescence in DP cells. Resveratrol (50, 100 μM) and plant extracts (400, 800 μg/ml) reversed PM2.5-induced cell senescence. Treatment with Res or EST significantly decreased SA-β-gal staining in PM2.5-treated cells. Furthermore, Res and EST decreased the mRNA levels of SASP, including IL1α, IL7, IL8, and CXCL1. DP cells exposed to PM2.5 exhibited an increase in p21 and p16 mRNA and protein levels, which could be reversed by the addition of Res or EST. Res and EST could reduce p21 and p16 in senescent cells approximately 3- and 2-fold, respectively, compared to untreated senescent cells. PM2.5 induced senescence in human DP stem cells. Res and EST extract potentially reverse the senescence phenotypes of such cells.

Amphipols (APol) are polymers which can solubilise and stabilise membrane proteins (MP) in aqueous solutions. In contrast to conventional detergents, APol are able to keep MP soluble even when the free APol concentration is very low. Outer membrane protein F (OmpF) is the most abundant MP commonly found in the outer membrane (OM) of Escherichia coli . It plays a vital role in the transport of hydrophilic nutrients, as well as antibiotics, across the OM. In the present study, APol was used to solubilise OmpF to characterize its interactions with molecules such as lipopolysaccharides (LPS) or colicins. OmpF was reconstituted into APol by the removal of detergents using Bio-Beads followed by size-exclusion chromatography (SEC) to remove excess APol. OmpF/APol complexes were then analysed by SEC, dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM showed that in the absence of free APol–OmpF associated as long filaments with a thickness of ~6 nm. This indicates that the OmpF trimers lie on their sides on the carbon EM grid and that they also favour side by side association. The formation of filaments requires APol and occurs very rapidly. Addition of LPS to OmpF/APol complexes impeded filament formation and the trimers form 2D sheets which mimic the OM. Consequently, free APol is undoubtedly required to maintain the homogeneity of OmpF in solutions, but ‘minimum APol’ provides a new phase, which can allow weaker protein–protein and protein–lipid interactions characteristic of native membranes to take place and thus control 1D–2D crystallisation.

Gram-negative bacteria are becoming increasingly resistant to many antibiotics. Unlike other bacteria, gram-negative bacteria have an additional membrane, the so-called outer membrane, which protects them from harmful agents. This membrane is highly asymmetric and contains tightly-packed lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet. This thesis describes a study of the outer membrane protein F (OmpF) which is the most abundant porin present in the outer membrane of Esherichia coli. OmpF plays a key role in the organization of outer membrane and is also a receptor and translocator for colicins, which are antibacterial toxins. The aim of this project is to better understand the defensive barrier of gram-negative bacteria and find routes to pass through it by studying the interaction of OmpF with colicin N (ColN) and LPS. The solution structure of ColN and the OmpFColN complex in neutral detergents, studied by AUC and small-angle scattering (SAS), indicated that translocation and receptor binding domain of ColN (ColNTR) became more compact when binding to outside of OmpF. Furthermore, OmpF in complex with the protein TolA, which forms part of the ColN translocon complex, was also studied by SAS. It showed that TolA bound to OmpF also became more compact though TolA remains a flexible structure. Furthermore, the interaction of OmpF with LPS was studied. Mutagenesis of positivelycharged residues on OmpF was used to disrupt its electrostatic interaction with LPS. The findings suggested that OmpF has two LPS-binding sites and that OmpF binds to LPS via the minimal Lipid A moiety. This was supported by SAS data. Dynamic light scattering experiments indicated that OmpF, LPS and divalent cations form larger-scale structures, which are reminiscent of the outer membrane of bacteria. Alternative approaches for studying OmpF in amphipol (APol) and nanodisc were also utilised. OmpF/APol assembled as filaments in the absence of free APol but these converted into 2D arrays in the presence of LPS and calcium ions. OmpF could be incorporated into Nanodiscs, significantly increasing their diameters compared to empty Nanodiscs.