Using the Zebrafish Larval Model of Infection to Investigate Antibiotic Efficacy and Combination Treatments Against Staphylococcus aureus.

There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.

Therapeutic potential of Aloe vera-coated curcumin encapsulated nanoparticles in an Alzheimer-induced mice model: behavioural, biochemical and histopathological evidence.

OBJECTIVE: The main purpose of the present study was to evaluate the therapeutic efficacy of Aloe vera-coated curcumin encapsulated nanoparticles in mitigating Alzheimer's disease progression in mice, by examining behavioural changes, biochemical markers, and histopathological alterations, thus elucidating its potential as a treatment strategy. METHODS: The green synthesis method was used to synthesise this nanoformulation, which was then characterised using a variety of techniques, including percentage encapsulation efficacy, UV-visible spectroscopy, DLS, FT-IR, FESEM, and EDX. Several in-vivo assessments, including behavioural evaluations, dose optimisation studies, oxidative stress marker estimation, and histological studies, were conducted to determine the potential therapeutic impact of nanoformulation on the Alzheimer-induced mice model. RESULTS: The synthesised nanoparticles show a mean diameter of 76.12 nm ±1.23, a PDI of 0.313 ± 0.02, a zeta potential of 6.27 ± 0.65 mV, and the percentage encapsulation efficiency between 90% and 95% indicating good stability of synthesised nanoformulation. With the help of Morris water maze, Y-maze, and novel object recognition assay, the learning capacity and memory were assessed, and the results show that the synthesised nanoformulation significantly decreased the transfer latency to reach baited arm or to the hidden platform within 7 days. CONCLUSION: The formulation demonstrated significant biochemical benefits and remarkable cognitive advantages, establishing it as a prospective therapeutic intervention option that is both safe and effective.

Biological evaluation of novel phosphorylated pullulan-based calcium hydroxide formulations as direct pulp capping materials: An in vivo study on a rat model.

AIM: Calcium hydroxide (CH) has been considered as a direct pulp capping materials (DPC) for the last decades despite having some limitations. Phosphorylate pullulan (PPL) incorporated with CH (CHPPL) is a novel biomaterial that was introduced as a promising DPC material. Thus, the aim of the study was to evaluate the inflammatory response and mineralized tissue formation (MTF) ability of PPL-based CH formulations on rat molars after DPC. METHODOLOGY: This study consisted of six groups: CH with 1% PPL (CHPPL-1); 3% PPL (CHPPL-3); 5% PPL (CHPPL-5); Dycal and NEX MTA Cement (N-MTA) as the positive control, and no capping materials (NC). One hundred twenty maxillary first molar cavities were prepared on Wistar rats. After capping, all the cavities were restored with 4-META/MMA-TBB resin and pulpal responses were evaluated at days 1, 7, and 28. Kruskal-Wallis followed by Mann-Whitney U-test was performed with a significance level of 0.05. Immunohistochemical expression of IL-6, Nestin, and DMP-1 was observed. RESULTS: At day 1, CHPPL-1, N-MTA, and Dycal exhibited no to mild inflammation, whilst CHPPL-3, CHPPL-5, and NC showed mild to moderate inflammation, and the results were significantly different (p < .05). At day 7, mild to moderate inflammation was observed in CHPPL-1, N-MTA, and Dycal, whereas CHPPL-3, CHPPL-5, and NC exhibited moderate to severe inflammation. Significant differences were observed between CHPPL-1 and N-MTA with NC (p < .05), CHPPL-1 and CHPPL-3 with CHPPL-5 and Dycal (p < .05), and CHPPL-3 with N-MTA (p < .05). A thin layer of mineralized tissue formation (MTF) was observed in all groups. At day 28, CHPPL-1, Dycal, and N-MTA showed no to mild inflammation, whilst CHPPL-3, CHPPL-5, and NC exhibited mild to severe inflammation, and statistically significant difference was detected (p < .05). CHPPL-1, Dycal, and N-MTA exhibited continuous MTF, whilst CHPPL-3, CHPPL-5, and NC had thicker and interrupted MTF. Significant differences were observed between CHPPL-1, CHPPL-3, and N-MTA with NC group (p < .05). Variable expressions of IL-6, Nestin, and DMP-1 indicated differences in the materials' impact on odontoblast-like cell formation and tissue mineralization. CONCLUSIONS: These findings suggest that CHPPL-1 has the potential to minimize pulpal inflammation and promote MTF and had similar efficacy as MTA cement.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Ameliorative Effect of Natural Sesquiterpene Alcohol Cedrol Against Cerebral Ischemia Infarction-In Vitro and In Vivo Studies.

Cedrol is a major bioactive compound present in the Cedrus atlantica with numerous biological properties. In this study, we elucidated the neuroprotective properties of cedrol against ischemic infarction in animal and in vitro studies. A cerebral ischemic/reperfusion model was induced in adult Wistar rats, and oxygen-glucose deprivation/reperfusion was induced in SH-SY5Y neuronal cells and treated with different concentrations of cedrol. The percentage of water content, cerebral  infarct, and neurological deficit score was assessed in experimental rats. The acetylcholinesterase activity and inflammatory cytokines were quantified to analyze the anti-inflammatory potency of cedrol. Oxidative stress marker malondialdehyde and antioxidants were quantified to evaluate the antioxidant potency of cedrol in an ischemic condition. The neuroprotective potency of cedrol was confirmed by histopathological analysis of the brain tissue of cedrol-treated I/R-induced rats. In in vitro studies, the MTT and LDH assays were performed in cedrol-treated OGD/R SH-SY5Y cells to analyze the cytoprotective effect of cedrol. The anti-inflammatory property of cedrol was confirmed by quantifying the pro-inflammatory cytokine levels in OGD/R-induced cedrol-treated SH-SY5Y cells. The results obtained prove that cedrol significantly prevents brain edema, neurological deficits, acetylcholinesterase activity, and oxidative damage in ischemic-induced rats. It inhibited neuroinflammation in ischemic-induced rats and also in in vitro models. The neuroprotective effect of cedrol during an ischemic condition was authentically established with histological analysis in an animal model and cell survival assays in an in vitro model. Overall, our results confirm that cedrol is a potent alternative drug to treat cerebral ischemia in the future.

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line.

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

Emerging In Vitro and In Vivo Models: Hope for the Better Understanding of Cancer Progression and Treatment.

Various cancer models have been developed to aid the understanding of the underlying mechanisms of tumor development and evaluate the effectiveness of various anticancer drugs in preclinical studies. These models accurately reproduce the critical stages of tumor initiation and development to mimic the tumor microenvironment better. Using these models for target validation, tumor response evaluation, resistance modeling, and toxicity comprehension can significantly enhance the drug development process. Herein, various in vivo or animal models are presented, typically consisting of several mice and in vitro models ranging in complexity from transwell models to spheroids and CRISPR-Cas9 technologies. While in vitro models have been used for decades and dominate the early stages of drug development, they are still limited primary to simplistic tests based on testing on a single cell type cultivated in Petri dishes. Recent advancements in developing new cancer therapies necessitate the generation of complicated animal models that accurately mimic the tumor's complexity and microenvironment. Mice make effective tumor models as they are affordable, have a short reproductive cycle, exhibit rapid tumor growth, and are simple to manipulate genetically. Human cancer mouse models are crucial to understanding the neoplastic process and basic and clinical research improvements. The following review summarizes different in vitro and in vivo metastasis models, their advantages and disadvantages, and their ability to serve as a model for cancer research.

Orthotopic model for the analysis of melanoma circulating tumor cells.

Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.

Using the model cestode Taenia crassiceps for the study of cysticercosis.

Taenia solium is the aetiological agent of taeniasis/cysticercosis, one of the most severe neglected tropical diseases (NTD) according to the World Health Organization (WHO). The life cycle of T. solium alternates between pigs (intermediate host) and humans (definitive host). In addition, humans can act as accidental intermediate hosts if they ingest infective eggs. In this case, the most severe condition of the disease occurs when parasites invade the central nervous system, causing neurocysticercosis (NCC). The complexity of the life cycle of T. solium imposes a barrier to study this pathogen thoroughly. Thus, related species, such as T. crassiceps are commonly used. Due to its capacity to multiply asexually, T. crassiceps can be maintained by serial passage in laboratory mice in standard biosecurity level facilities. In addition, an in vitro system to generate cysticerci in the presence of feeder cells has been recently developed. Despite model species display biological differences with their zoonotic counterparts, they have historically helped to understand the biology of the related pathogenic species and hence, generate improvements in NTD detection and control. In this chapter, we describe the procedures to carry out both in vivo and in vitro systems for T. crassiceps in the laboratory.

Blue light protection factor: a method to assess the protective efficacy of cosmetics against blue light-induced skin damage in the Chinese population.

BACKGROUND: Previous studies have shown that visible light (VL), especially blue light (BL), could cause significant skin damage. With the emergence of VL protection products, a harmonization of light protection methods has been proposed, but it has not been widely applied in the Chinese population. OBJECTIVE: Based on this framework, we propose an accurate and simplified method to evaluate the efficacy of BL photoprotection for the Chinese population. METHODS: All subjects (n = 30) were irradiated daily using a blue LED light for four consecutive days. Each irradiation dose was 3/4 MPPD (minimum persistent pigmentation darkening). The skin pigmentation parameters, including L*, M, and ITA°, were recorded. We proposed the blue light protection factor (BPF) metric based on the skin pigmentation parameters to evaluate the anti-blue light efficacies of different products. RESULTS: We found that the level of pigmentation rose progressively and linearly as blue light exposure increased. We proposed a metric, BPF, to reflect the anti-blue light efficacy of products based on the linear changes in skin pigment characteristics following daily BL exposure. Moreover, we discovered that the BPF metric could clearly distinguish the anti-blue light efficacies between two products and the control group, suggesting that BPF is an efficient and simple-to-use metric for anti-blue light evaluation. CONCLUSION: Our study proposed an accurate and simplified method with an easy-to-use metric, BPF, to accurately characterize the anti-blue light efficacies of cosmetic products, providing support for further development of anti-blue light cosmetics.

Continuous iontronic chemotherapy reduces brain tumor growth in embryonic avian in vivo models.

Local and long-lasting administration of potent chemotherapeutics is a promising therapeutic intervention to increase the efficiency of chemotherapy of hard-to-treat tumors such as the most lethal brain tumors, glioblastomas (GBM). However, despite high toxicity for GBM cells, potent chemotherapeutics such as gemcitabine (Gem) cannot be widely implemented as they do not efficiently cross the blood brain barrier (BBB). As an alternative method for continuous administration of Gem, we here operate freestanding iontronic pumps - "GemIPs" - equipped with a custom-synthesized ion exchange membrane (IEM) to treat a GBM tumor in an avian embryonic in vivo system. We compare GemIP treatment effects with a topical metronomic treatment and observe that a remarkable growth inhibition was only achieved with steady dosing via GemIPs. Daily topical drug administration (at the maximum dosage that was not lethal for the embryonic host organism) did not decrease tumor sizes, while both treatment regimes caused S-phase cell cycle arrest and apoptosis. We hypothesize that the pharmacodynamic effects generate different intratumoral drug concentration profiles for each technique, which causes this difference in outcome. We created a digital model of the experiment, which proposes a fast decay in the local drug concentration for the topical daily treatment, but a long-lasting high local concentration of Gem close to the tumor area with GemIPs. Continuous chemotherapy with iontronic devices opens new possibilities in cancer treatment: the long-lasting and highly local dosing of clinically available, potent chemotherapeutics to greatly enhance treatment efficiency without systemic side-effects. SIGNIFICANCE STATEMENT: Iontronic pumps (GemIPs) provide continuous and localized administration of the chemotherapeutic gemcitabine (Gem) for treating glioblastoma in vivo. By generating high and constant drug concentrations near the vascularized growing tumor, GemIPs offer an efficient and less harmful alternative to systemic administration. Continuous GemIP dosing resulted in remarkable growth inhibition, superior to daily topical Gem application at higher doses. Our digital modelling shows the advantages of iontronic chemotherapy in overcoming limitations of burst release and transient concentration profiles, and providing precise control over dosing profiles and local distribution. This technology holds promise for future implants, could revolutionize treatment strategies, and offers a new platform for studying the influence of timing and dosing dependencies of already-established drugs in the fight against hard-to-treat tumors.

Zophobas morio larvae as a novel model for the study of Acinetobacter virulence and antimicrobial resistance.

The use of mammalian models for in vivo testing of bacterial virulence raises ethical concerns and is expensive and time-consuming. As an alternative, non-mammalian models are sought. Galleria mellonella larvae have been used as a model to study several bacterial pathogens. However, their maintenance is challenging, and commercial supply is low. In this study, we aimed to establish the Zophobas morio larvae as an alternative non-mammalian model for the evaluation of the pathogenicity and antimicrobial susceptibility of Acinetobacter baumannii. We infected Z. morio with Acinetobacter strains and determined the optimal temperature and inoculum. To visualize the bacterial distribution within the larvae, hematoxylin and eosin (H&E) staining was performed. Next, a survival model of infected larvae was established, and virulence was compared between strains. The effect of antimicrobial treatment in relation to antibiotic susceptibility was studied. Our results demonstrate that Z. morio can be used as a model system for in vivo studies of A. baumannii.

Method for evaluating milk production in mouse mammary gland.

Lactation is a characteristic physiological function of mammals and is important for nourishing infants and the dairy industry; however, the molecular mechanisms underlying the function remain to be elucidated. A technique to directly evaluate the quantity and quality of milk in mice is necessary for the study of the lactation mechanism in vivo. By measuring the changes in milk amount after different durations of milk accumulation (0-24 h) using a ductal cannulation technique and oxytocin supplementation, we estimated the milk production rate at a single mammary gland level. In addition, collected milk was available to assess milk quality, including creamatocrit, osmolarity, and concentrations of ions, lactose, and total protein. Moreover, as a proof of principle, the effects of intraductal administration of a hypertonic solution to the abdominal mammary gland were examined. This stimulation increased milk amount, possibly by osmosis, compared with the contralateral control gland. These results demonstrated that this method is useful for examining the lactation ability and mechanisms in vivo. Studies using this method will contribute to the further understanding of lactation mechanisms in mammals.

Laherradurin Inhibits Tumor Growth in an Azoxymethane/Dextran Sulfate Sodium Colorectal Cancer Model In Vivo.

Colorectal cancer (CRC) is the third most common neoplasia in the world. Its mortality rate is high due to the lack of specific and effective treatments, metastasis, and resistance to chemotherapy, among other factors. The natural products in cancer are a primary source of bioactive molecules. In this research, we evaluated the antitumor activity of an acetogenin (ACG), laherradurin (LH), isolated from the Mexican medicinal plant Annona macroprophyllata Donn.Sm. in a CRC murine model. The CRC was induced by azoxymethane-dextran sulfate sodium (AOM/DSS) in Balb/c mice and treated for 21 days with LH or cisplatin. This study shows for the first time the antitumor activity of LH in an AOM/DSS CRC model. The acetogenin diminished the number and size of tumors compared with cisplatin; the histologic studies revealed a recovery of the colon tissue, and the blood toxicity data pointed to less damage in animals treated with LH. The TUNEL assay indicated cell death by apoptosis, and the in vitro studies exhibited that LH inhibited cell migration in HCT116 cells. Our study provides strong evidence of a possible anticancer agent for CRC.

Intra-articular injection of platelet lysate-derived extracellular vesicles recovers from knee osteoarthritis in an in vivo rat model.

Objective: MSCs and Platelet-Rich Plasma are the main focus in the study of new regenerative treatments aimed to reverse Osteoarthritis (OA). However, extracellular vesicles (EVs) present several advantages to cell-based treatments. Thus, the aim of this study was to compare and evaluate the regenerative potential of MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in an OA cartilage rat model. Design: OA in vivo model was established through injection of 6 mg MIA in the rat knee joints. After 14 and 21 days, OA knee joints were treated with 1 × 1010 particles of pEVs or cEVs. At day 28, the animals were sacrificed, plasma was collected to quantify CTX-II and knee joints were excised to be evaluated by Cone Beam Computed Tomography (CBCT). After decalcification, histology was used to determine the OARSI score and to visualize collagen and glycosaminoglycan content. Results: pEVs and cEVs samples did not show significant differences per se but they did in terms of regenerative effects on OA knee joints. pEVs-treated knee joints showed better subchondral bone integrity in CT-analysed parameters when compared to cEVs or OA group, showing similar values to the healthy control group. Moreover, OARSI score indicated that pEVs showed a greater OA reversion in knee joints, especially in female rats, and so indicated the analysed histological images. Conclusions: pEVs are proposed as a viable regeneration treatment for OA since they are not only capable of exerting their regenerative potential on osteoarthritic cartilage, but also outperform cEVs in terms of efficacy, particularly in females. Significance statement: Osteoarthritis (OA) is one of the most age-related diseases. It is estimated that 500 million people suffer from OA worldwide, representing the principal cause of chronic disability in adults. In the present study we evaluated the therapeutic effect of extracellular vesicles (EVs) from different sources (platelet lysate and human umbilical cord mesenchymal stromal cells) in an in vivo rat model. Our results demonstrate that platelet-derived EVs (pEVs) induce an OA reversion in knee joints, thus evidencing the therapeutic potential of pEVs as cell-free regenerative agents for OA treatment. The translational potential of this article: Platelet-derived extracellular vesicles (pEVs) offer a promising cell-free therapy option for OA treatment. Their production could be easily standardized and reproduced without extensive platelet harvesting and amplification, thus paving the way for their clinical translation.

Nickel-doped vanadium pentoxide (Ni@V2O5) nanocomposite induces apoptosis targeting PI3K/AKT/mTOR signaling pathway in skin cancer: An in vitro and in vivo study.

In the present study, the vanadium pentoxide (V2O5) nickel-doped vanadium pentoxide (Ni@V2O5) was prepared and determined for in vitro anticancer activity. The structural characterization of the prepared V2O5 and Ni@V2O5 was determined using diverse morphological and spectroscopic analyses. The DRS-UV analysis displayed the absorbance at 215 nm for V2O5 and 331 nm for Ni@V2O5 as the primary validation of the synthesis of V2O5 and Ni@V2O5. The EDS spectra exhibited the presence of 30% of O, 69% of V, and 1% of Ni and the EDS mapping showed the constant dispersion. The FE-SEM and FE-TEM analysis showed the V2O5 nanoparticles are rectangle-shaped and nanocomposites have excellent interfaces between nickel and V2O5. The X-ray photoelectron spectroscopy (XPS) investigation of Ni@V2O5 nanocomposite endorses the occurrence of elements V, O, and Ni. The in vitro MTT assay clearly showed that the V2O5 and Ni@V2O5 have significantly inhibited the proliferation of B16F10 skin cancer cells. In addition, the nanocomposite produces the endogenous reactive oxygen species in the mitochondria, causes the mitochondrial membrane and nuclear damage, and consequently induces apoptosis by caspase 9/3 enzymatic activity in skin cancer cells. Also, the western blot analysis showed that the nanocomposite suppresses the oncogenic marker proteins such as PI3K, Akt, and mTOR in the skin cancer cells. Together, the results showed that Ni@V2O5 can be used as an auspicious anticancer agent against skin cancer.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

Stroma modulation of radiation response in head and neck squamous cell carcinoma: Insights from zebrafish larvae xenografts.

BACKGROUND: The tumour microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) consists of different subtypes of cells that interact with the tumour or with each other. This study investigates the possibility of co-culturing HNSCC cells with different stroma cells in a zebrafish xenograft model, focusing on the effect of stroma cells on HNSCC growth and response to irradiation. MATERIAL AND METHOD: HNSCC metastatic cell line HSC-3 was used along with five types of stroma cells: normal gingival fibroblasts (NOF), cancer associated fibroblasts (CAF), macrophages, CD4+ T cells, and human umbilical vein endothelial cells (HUVEC). The mixture of HSC-3 cells and each-stroma cell type-was injected into 2-day post-fertilization zebrafish embryos, and the effect of stroma cells on tumour growth was tested. The study also aimed to mimic the HNSCC tumour by injecting a mixture of HSC-3 cells, CAFs, macrophages, and HUVECs into zebrafish embryos and testing the effect of these stroma cells on the cancer cells' response to irradiation compared to HSC-3-only tumours. RESULTS: CAFs had a significant inducement effect on tumour size, while HUVECs showed the opposite effect. The irradiated group of HSC-3-only tumour had a significantly smaller tumor cell area compared to the control, while the group with stroma cells and HSC-3 cells showed cancer cells being resistant to irradiation. CONCLUSION: This is the first report of co-culturing cancer cells with several types of stroma cells using a zebrafish xenograft model. This study also highlighted the role of stroma cells in turning the cancer cells from radioresponsive to radioresistant.

Ovine model of congenital chest wall and spine deformity: From birth to 3 months follow-up.

Background: The evolution and treatment of lung alterations related to congenital spine and chest wall deformities (CWD) are poorly understood. Most animal models of CWD created postnatally were not evaluated for respiratory function. The goal of our study was to evaluate the effects of a CWD induced in utero on lung growth and function in an ovine model. Methods: A CWD was induced in utero at 70-75 days of gestation in 14 ovine fetuses by resection of the 7th and 8th left ribs. Each non-operated twin fetus was taken as control. Respiratory mechanics was studied postnatally in the first week and at 1, 2, and 3 months. Post-mortem respiratory mechanics and lung histomorphometry were also assessed at 3 months. Results: Eight out of 14 CWD lambs (57%) and 14 control lambs survived the postnatal period. One severe and five mild deformities were induced. At birth, inspiratory capacity (25 vs. 32 mL/kg in controls), and dynamic (1.4 vs. 1.8 mL/cmH2O/kg), and static (2.0 vs. 2.5 mL/cmH2O/kg) respiratory system compliances were decreased in CWD lambs. Apart from a slight decrease in inspiratory capacity at 1 month of life, no other differences were observed in respiratory mechanics measured in vivo thereafter. Postmortem measurements found a significant decrease in lung compliance-for each lung and for both lungs taken together-in CWD lambs. No differences in lung histology were detected at 3 months in CWD animals compared to controls. Conclusions: Our study is the first to assess the effects of a prenatally induced CWD on lung development and function from birth to 3 months in an ovine model. Our results show no significant differences in lung histomorphometry at 3 months in CWD lambs compared to controls. Resolution at 1 month of the alterations in respiratory mechanics present at birth may be related to the challenge in inducing severe deformities.