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Scientific Reports Mar 2024Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current...
Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.
Topics: Humans; Enterotoxins; Bacterial Toxins; Caco-2 Cells; Melitten; Nigericin; Intestinal Mucosa
PubMed: 38461178
DOI: 10.1038/s41598-024-56520-5 -
Iranian Biomedical Journal Jan 2024The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the...
BACKGROUND
The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the UPR and autophagy as two critical pathways involved in tumorigenesis of colorectal and drug resistance. This study aimed to investigate the effect of melittin on these pathways in the colorectal cancer (CRC) HCT116 cells.
METHODS
MTT method was carried out to assess the cytotoxicity of melittin on the HCT116 cell line for 24, 48, and 72 h. After selecting the optimal concentrations and treatment times, the gene expression of autophagy flux markers (LC3-βII and P62) and UPR markers (CHOP and XBP-1s) were determined using qRT-PCR. The protein level of autophagy initiation marker (Beclin1) was also determined by Western blotting.
RESULTS
MTT assay showed a cytotoxic effect of melittin on the HCT116 cells. The increase in LC3-βII and decrease in P62 mRNA expression levels, along with the elevation in the Beclin1 protein level, indicated the stimulatory role of melittin on the autophagy. Melittin also significantly enhanced the CHOP and XBP-1s expressions at mRNA level, suggesting the positive role of the melittin on the UPR activation.
CONCLUSION
This study shows that UPR and autophagy can potentially be considered as two key signaling pathways in tumorigenesis, which can be targeted by the BV melittin in the HCT116 cells. Further in vivo evaluations are recommended to verify the obtained results.
Topics: Humans; HCT116 Cells; Melitten; Beclin-1; Unfolded Protein Response; Autophagy; RNA, Messenger; Carcinogenesis; Colorectal Neoplasms
PubMed: 38445441
DOI: 10.61186/ibj.3993 -
Toxicology Mar 2024The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints...
The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated. Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.
Topics: Bee Venoms; DNA; DNA Damage; Mitochondria; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Superoxides; Transcription Factors; Humans
PubMed: 38442839
DOI: 10.1016/j.tox.2024.153768 -
Dental and Medical Problems 2024Diabetes mellitus (DM) is a critical chronic metabolic disease. Several treatment modalities are currently under investigation. Both bee venom (BV) and bone marrow...
BACKGROUND
Diabetes mellitus (DM) is a critical chronic metabolic disease. Several treatment modalities are currently under investigation. Both bee venom (BV) and bone marrow mesenchymal stem cells (BMSCs) can possibly offer an approach for treating type I diabetes.
OBJECTIVES
The aim of the present study was to investigate the mechanism underlying the anti-diabetic effect of BV as compared to BMSCs on the tongue mucosa of diabetic rats.
MATERIAL AND METHODS
A total of 52 male albino rats were used in the current study. The rats were randomly assigned into 4 groups: group 1 (control); group 2 (streptozocin (STZ)); group 3 (BV-treated); and group 4 (BMSC-treated). Diabetes mellitus was induced via an intraperitoneal (IP) injection of STZ in the rats from groups 2, 3 and 4. Following the diagnosis of DM, the rats in group 3 were injected with a daily dose of 0.5 mg/kg of BV, while the rats in group 4 were treated with a single injection of BMSCs. All rats were euthanized after 4 weeks, and their tongues were dissected and divided into halves. The right halves of the tongues were utilized for the histological examination, followed by morphometric analysis. In contrast, the left halves were used to detect the local gene expression of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF).
RESULTS
Group 2 revealed marked disruption in the morphology of the fungiform and filiform papillae, and atrophic epithelial changes in both dorsal and ventral surface epithelium as compared to other groups. Group 4 showed a significantly larger number of taste buds, and a higher gene expression of TGF-β1 and VEGF as compared to groups 2 and 3. Additionally, BV and BMSCs effectively increased the thickness of dorsal and ventral surface epithelium as compared to group 2.
CONCLUSIONS
Treatment with BMSCs was associated with significant improvement in the morphology and number of lingual epithelial cells and taste buds in the tongues of diabetic rats as compared to BV-treated rats, which was due to the local upregulation of TGF-β1 and VEGF gene expression.
Topics: Male; Animals; Rats; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Diabetes Mellitus, Experimental; Tongue; Mesenchymal Stem Cells; Bee Venoms
PubMed: 38441304
DOI: 10.17219/dmp/152924 -
Scientific Reports Feb 2024The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na channels (VGNCs) and slows their...
The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Na1.2 and Na1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na influx in the AIS. Finally, using ultrafast Ca imaging, we observed that 7 nM AaH-II produces a spurious slow Ca influx via Ca-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.
Topics: Mice; Animals; Action Potentials; Axon Initial Segment; Scorpions; Peptides; Scorpion Venoms; Animals, Poisonous
PubMed: 38424206
DOI: 10.1038/s41598-024-55315-y -
Nano Letters Mar 2024Artificial organelles (AnOs) are in the spotlight as systems to supplement biochemical pathways in cells. While polymersome-based artificial organelles containing...
Artificial organelles (AnOs) are in the spotlight as systems to supplement biochemical pathways in cells. While polymersome-based artificial organelles containing enzymes to reduce reactive oxygen species (ROS) are known, applications requiring control of their enzymatic activity and cell-targeting to promote intracellular ROS detoxification are underexplored. Here, we introduce advanced AnOs where the chemical composition of the membrane supports the insertion of pore-forming melittin, enabling molecular exchange between the AnO cavity and the environment, while the encapsulated lactoperoxidase (LPO) maintains its catalytic function. We show that HO outside AnOs penetrates through the melittin pores and is rapidly degraded by the encapsulated enzyme. As surface attachment of cell-penetrating peptides facilitates AnOs uptake by cells, electron spin resonance revealed a remarkable enhancement in intracellular ROS detoxification by these cell-targeted AnOs compared to nontargeted AnOs, thereby opening new avenues for a significant reduction of oxidative stress in cells.
Topics: Reactive Oxygen Species; Artificial Cells; Hydrogen Peroxide; Melitten; Oxidative Stress
PubMed: 38408754
DOI: 10.1021/acs.nanolett.3c03888 -
PloS One 2024Scorpion venoms are known to contain over 100,000 biologically active constituents. However, only a few of them have been studied. The major constituents of venom are...
Scorpion venoms are known to contain over 100,000 biologically active constituents. However, only a few of them have been studied. The major constituents of venom are proteins and peptides, which exhibit various biological and pharmacological properties, including anticancer activities. In the current study, the venom of yellow scorpions (Buthus sindicus) found in Sindh, Pakistan, was extracted and evaluated for its anti-cancer and anti-inflammatory activities. The crude venom showed a dose dependent inhibition of phagocyte oxidative burst from human whole blood cells (28.3% inhibition at highest tested concentration of 300 μg/mL). In-vitro cytotoxicity of crude venom was evaluated against human prostrate (PC3), cervical (HeLa) and neuroblastoma (U87-MG) cell lines, along with cytotoxicity against normal human fibroblast (BJ) cells. Crude venom was cytotoxic to all cell lines, with prominent inhibitory effect on PC3 cells. Crude venom was fractionated through RP-UPLC, resulted in fifteen fractions, followed by evaluation of their anticancer potential. Among all, the fraction I significantly (P < 0.001) reduced the cell viability of all three cancer cell lines, and exhibited insignificant cytotoxicity against normal cell line. Furthermore, the apoptotic cell death pathway was evaluated for crude venom, and fraction I, in most sensitive cell line PC3, by using flow-cytometry analysis. Both crude venom and its fraction I caused a mitochondrial-mediated apoptosis in prostate cancer cells (PC3). To the best of our knowledge, this is the first report of the anticancer and anti-inflammatory activity of venom of Pakistani yellow scorpions. Results indicate their therapeutic potential, and a case for further purification and validation studies.
Topics: Male; Animals; Humans; Scorpions; Prostate; Peptides; Apoptosis; Cell Line, Tumor; Brain; Anti-Inflammatory Agents; Scorpion Venoms
PubMed: 38394321
DOI: 10.1371/journal.pone.0296636 -
Toxins Feb 2024Limited evidence suggests that stimulating adipose-derived stem cells (ASCs) indirectly promotes hair growth. We examined whether bee venom (BV) activated ASCs and...
Limited evidence suggests that stimulating adipose-derived stem cells (ASCs) indirectly promotes hair growth. We examined whether bee venom (BV) activated ASCs and whether BV-induced hair growth was facilitated by enhanced growth factor release by ASCs. The induction of the telogen-to-anagen phase was studied in mice. The underlying mechanism was investigated using organ cultures of mouse vibrissa hair follicles. When BV-treated ASCs were injected subcutaneously into mice, the telogen-to-anagen transition was accelerated and, by day 14, the hair weight increased. Quantitative polymerase chain reaction (qPCR) revealed that BV influenced the expression of several molecules, including growth factors, chemokines, channels, transcription factors, and enzymes. Western blot analysis was employed to verify the protein expression levels of extracellular-signal-regulated kinase (ERK) and phospho-ERK. Both the Boyden chamber experiment and scratch assay confirmed the upregulation of cell migration by BV. Additionally, ASCs secreted higher levels of growth factors after exposure to BV. Following BV therapy, the gene expression levels of alkaline phosphatase (ALP), fibroblast growth factor (FGF)-1 and 6, endothelial cell growth factor, and platelet-derived growth factor (PDGF)-C were upregulated. The findings of this study suggest that bee venom can potentially be utilized as an ASC-preconditioning agent for hair regeneration.
Topics: Animals; Mice; Bee Venoms; Cell Proliferation; Hair; Intercellular Signaling Peptides and Proteins; Stem Cells; Cells, Cultured
PubMed: 38393162
DOI: 10.3390/toxins16020084 -
Protein Science : a Publication of the... Mar 2024Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and...
Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and developing broadly-neutralizing antibodies can be cumbersome using traditional low-throughput iterative protein engineering methods. Here, we present a new high-throughput approach for the standardized discovery of broadly-neutralizing monoclonal antibodies relying on phage display technology and consensus antigens representing average sequences of related proteins. We showcase the utility of this approach by applying it to toxic sphingomyelinases from the venoms of species from very distant orders of the animal kingdom, the recluse spider and Gadim scorpion. First, we designed a consensus sphingomyelinase and performed three rounds of phage display selection, followed by DELFIA-based screening and ranking, and benchmarked this to a similar campaign involving cross-panning against recombinant versions of the native toxins. Second, we identified two scFvs that not only bind the consensus toxins, but which can also neutralize sphingomyelinase activity of native whole venom in vitro. Finally, we conclude that the phage display campaign involving the use of the consensus toxin was more successful in yielding cross-neutralizing scFvs than the phage display campaign involving cross-panning.
Topics: Animals; Sphingomyelin Phosphodiesterase; Brown Recluse Spider; Scorpions; Broadly Neutralizing Antibodies; Consensus; Antibodies, Monoclonal; Spider Venoms
PubMed: 38358130
DOI: 10.1002/pro.4901