Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

Resveratrol Shows Potent Senescence Reversal in Experimental Cellular Models of Particular Matter 2.5-induced Cellular Senescence in Human Dermal Papilla Cells.

BACKGROUND/AIM: 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. MATERIALS AND METHODS: Cell senescence was determined using the ß-Galactosidase (SA-ß-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. RESULTS: 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-ß-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. CONCLUSION: PM2.5 induced senescence in human DP stem cells. Res and EST extract potentially reverse the senescence phenotypes of such cells.

The Role of Box A of HMGB1 in Enhancing Stem Cell Properties of Human Mesenchymal Cells: A Novel Approach for the Pursuit of Anti-aging Therapy.

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.

CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

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 IgG1 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 (IgG1/IgG2a) 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.

In Vitro Screening for Cytotoxic Effect of Pore Forming Colicin N and Its Domains on Human Cancer Cells.

Protein-based drugs have increasingly become an important segment of cancer treatment. In comparison with chemotherapy, they offer high efficacy and fewer side effects due to specifically targeting only cancer cells. Monoclonal antibodies are currently the main protein-based drugs in the market but their complexity and limitations in tumour penetration led to the development of alternative protein therapeutics such as pore-forming toxins. Colicin N (ColN), a pore-forming protein produced by E. coli, was previously found to exhibit cytotoxicity and selectivity in human lung cancer cells with promising potential for further development. Here we aimed to screen for the cytotoxicity of ColN in breast (MCF-7 and MDA-MB-231), lung (A549) and colon cancer cells (HT-29 and HCT-116) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with various concentrations for 72 h and to investigate the cytotoxic effect of ColN domains on cancer cells. It showed that ColN mildly mediated the decrease in cell viability except for MCF-7. The highest effect was seen in A549 and HCT-116 cells which showed 31.9% and 31.5% decrease in cell viability, respectively. The mild inhibition or promotion of cancer cell proliferation by ColN tend to be based on the cell types. Furthermore, to search for the functional domain of ColN used for cytotoxicity, full-length ColN and truncated ColN with deletion of translocating, receptor binding and pore-forming domains were also tested on HCT-116 colon cancer cells. The findings indicated that HCT-116 cells were not significantly sensitive to ColN but full length ColN caused slight decrease in cancer cell viability. The data in this study will benefit the further development of ColN for alternative protein-based cancer therapy.

Resurfacing receptor binding domain of Colicin N to enhance its cytotoxic effect on human lung cancer cells.

Colicin N (ColN) is a bacteriocin secreted by Escherichia coli (E. coli) to kill other Gram-negative bacteria by forcefully generating ion channels in the inner membrane. In addition to its bactericidal activity, ColN have been reported to selectively induce apoptosis in human lung cancer cells via the suppression of integrin modulated survival pathway. However, ColN showed mild toxicity against human lung cancer cells which could be improved for further applications. The protein resurfacing strategy was chosen to engineer ColN by extensive mutagenesis at solvent--exposed residues on ColN. The highly accessible Asp and Glu on wild-type ColN (ColNWT) were replaced by Lys to create polycationic ColN (ColN+12). Previous studies have shown that increase of positive charges on proteins leads to the enhancement of mammalian cell penetration as well as increased interaction with negatively charged surface of cancer cells. Those solvent--exposed residues of ColN were identified by Rosetta and AvNAPSA (Average number of Neighboring Atoms Per Side-chain Atom) approaches. The findings revealed that the structural features and stability of ColN+12 determined by circular dichroism were similar to ColNWT. Furthermore, the toxicity of ColN+12 was cancer -selective. Human lung cancer cells, H460 and H23, were sensitive to ColN but human dermal papilla cells were not. ColN+12 also showed more potent toxicity than ColNWT in cancer cells. This confirmed that polycationic resurfacing method has enabled us to improve the anticancer activity of ColN towards human lung cancer cells.

Colicin N Mediates Apoptosis and Suppresses Integrin-Modulated Survival in Human Lung Cancer Cells.

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 ß1 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.

Determining the amphipol distribution within membrane-protein fibre samples using small-angle neutron scattering.

Detergent micelles can solubilize membrane proteins, but there is always a need for a pool of free detergent at the critical micellar concentration to maintain the micelle-monomer equilibrium. Amphipol polymeric surfactants (APols) have been developed to replace conventional detergents in membrane-protein studies, but the role of free amphipol is unclear. It has previously been shown that the removal of free APol causes monodisperse outer membrane protein F (OmpF) to form long filaments. However, any remaining APol could not be resolved using electron microscopy. Here, small-angle neutron scattering with isotope contrast matching was used to separately determine the distributions of membrane protein and amphipol in a mixed sample. The data showed that after existing free amphipol had been removed from monodisperse complexes, a new equilibrium was established between protein-amphipol filaments and a pool of newly liberated free amphipol. The filaments consisted of OmpF proteins surrounded by a belt of Apol, whilst free oblate spheroid micelles of Apol were also present. No indications of long-range order were observed, suggesting a lack of defined structure in the filaments.

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis.

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.

An accurate in vitro model of the E.†coli envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

An Accurate In Vitro Model of the E.†coli Envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Outer membrane protein F stabilised with minimal amphipol forms linear arrays and LPS-dependent 2D crystals.

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.