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Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85–92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86–92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97–101%), akin to a Matrigel-based liver model (83–102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

In this study, the manufacture of zinc oxide nanoparticles (ZnO-NPs) was completed via the sol–gel method with Trigonella foenum-graecum L extract for the first time to function as the stabilizing and reducing agent. The obtained product was investigated by various analyzing procedures such as TGA/DTG, FT-IR, UV–Vis, XRD, and EDX/FESEM. The calcination of our product was conducted at temperatures of 400, 500, and 600 °C. In conformity to the XRD pattern, heightening the temperature of calcination caused an enlargement in the size of nanoparticles. The photocatalytic performance of ZnO-NPs was evaluated to degrade methylene blue and Eriochrome black T (EBT) dyes under UV light, which resulted in a degradation percentage of about 96% and 94%, after 90 min, respectively. There has been some evidence suggesting that the green synthesis of ZnO-NPs has increased their use in medicine. The outcomes of examining the cytotoxicity effect of this product against the Huh-7 cell line by the performance of the MTT assay were indicative of an IC 50 of around 62.5 µg/mL. Finally, according to the results of the broth microdilution method, which was performed to assess the antibacterial activity of ZnO-NPs towards gram-positive and gram-negative bacteria, the value of MIC was in the range of 31 to 125 µg/mL. The obtained results from biological studies confirm the antibacterial and anticancer properties of ZnO-NPs, which are promising for applying NPs in medical fields.

Nanoparticles have shown promising potential for efficient drug delivery, circumventing biological interferences like immunological and renal clearance and mechanical and enzymatic destruction. However, a handful of research papers have questioned the biomedical use of metal-based nanoparticles like cadmium telluride quantum dots (CdTe-QDs) for their cytotoxic, genotoxic, and carcinogenic potential. Herein, we examined the effects of CdTe-QD NPs on gene expression profile of hepatocellular carcinoma (Huh-7) cell line. Huh-7 cells were treated with CdTe-QD NPs (10 μg/ml for 6, 12, and 24 hours, and 25 μg/ml for 6 and 12 hours), and transcriptomic analysis was performed using microarray to evaluate the global gene expression profile. Differential expressed genes (DEGs) were observed for both the doses (10 and 25 μg/ml) of CdTe-QD NPs at different time points. Gene ontology (GO) analysis revealed that genes involved in molecular function of cell cycle, organizational injury and abnormalities, cell death and survival, gene expression, cancer, organismal survival, and cellular development were differentially expressed. Overall, we have demonstrated differential expression of several genes, involved in maintaining cell survival, metabolism, and genome integrity. These findings were confirmed by RT-qPCR study for some canonical pathway genes signifying possible implication in NP toxicity-mediated cell survival and inhibition of cell death.

INTRODUCTION: Evidence has shown that some microRNAs (miRNAs) play a role in tumorigenesis of hepatocellular carcinoma (HCC). Herein, we aimed to evaluate the diagnostic and prognostic values of serum exosomal miR-370-3p and miR-196a-5p in patients with HCC. MATERIAL AND METHODS: Serum exosomes in 90 HCC patients were extracted and identified. Serum exosomal miR-370-3p and miR-196a-5p expression in HCC patients were detected. The diagnostic value of miR-370-3p and miR-196a- 5p, relationship between miR-370-3p and miR-196a-5p expression and clinicopathological features and prognosis of patients with HCC were analyzed. Relationship between miR-370-3p and miR-196a-5p expression and liver function indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in HCC patients were analyzed. The effects of miR-370-3p and miR-196a-5p on Huh-7 HCC cells’ proliferation, invasion and migration were determined. RESULTS: Lower expression of miR-370-3p and higher expression of miR-196a-5p were found in serum exosomes of HCC patients. Serum exosomal miR-370-3p and miR-196a-5p were associated with tumor size, tumor grade and TNM stage as well as prognosis and liver function indices of HCC patients. Overexpressed miR-370-3p or silenced miR-196a-5p suppressed proliferation, invasion and migration of Huh-7 HCC cells. CONCLUSIONS: We suggest that miR-370-3p/miR-196a-5p in serum exosomes of HCC patients could be potential biomarkers for the diagnosis and prognosis of HCC.

Five new compounds called Pestalotis A–E (1–5), comprising three monoterpene-lactone compounds (1–3), one tetrahydrobenzofuran derivative (4), and one sesquiterpene (5), were isolated from the EtOAc extract of Pestalotiopsis sp. The structures of the new compounds were elucidated by analysis of their NMR, HRMS, and ECD spectra, and the absolute configurations were established through the comparison of experimental and calculated ECD spectra. All compounds were tested for antitumor activity against SW-480, LoVo, HuH-7, and MCF-7. The results showed that compounds 2 and 4 exhibited potent antitumor activity against SW-480, LoVo, and HuH-7 cell lines. Furthermore, compound 4 was assessed against HuH-7, and the results indicated that the rate of apoptosis was dose-dependent.

[...]for a wide range of biology applications, it is important to culture the cell for long periods of time[1]. Many reports have demonstrated that the micro-space cell culture plate method, a three-dimensional (3D) culture system, can induce hepatocyte-specific functions, including CYP2E1 expression, in HepG2 cells, which results in an increase in cytotoxicity by acetaminophen compared with a conventional two-dimensional (2D) culture system [16]. [...]it remains to be demonstrated whether cell injury induced by APAP in 3D-cultured HCC in vitro was produced by corresponding human and rodent mechanisms of in vivo APAP-induced hepatotoxicity and if antidotes such as N-acetylcysteine (NAC) or other agents also show protective potential against APAP-induced hepatotoxicity in the 3D-cultivation system [18-19]. Scaffolds were fixed in 4% paraformaldehyde for ten minutes at room temperature after culturing, at day 3 and day 7 followed by permeabilization with 0.5% TritonX-100 for 10min at 37°C. Actin filaments were stained with rhodamine-phalloidin (Molecular Probes) for 20min at 37 °C, and Nucleus was stained with DAPI (4,6-diamidino-2-phenylindole, dihydrochloride) (Molecular Probes) for 10min at 37°C. Images were acquired with a TCS SP5 II confocal microscope.

Background Hepatitis C virus (HCV) is estimated to infect 200 million individuals in the globe, including approximately 10 million in Pakistan causing both acute and chronic hepatitis. The standard treatment against HCV is pegylated interferon therapy in combination with a nucleoside analogue ribavirin. In addition, several herbal extracts and phytochemicals derivatives are used traditionally in the treatment of liver diseases as well as HCV infection. The present study determines the inhibitory effect of kaolin minerals compound against hepatitis C virus in Huh-7 cell lines . Methods Huh-7 cell lines were used for the in vitro HCV replication by using HCV positive sera from different patients with known HCV genotypes and viral titer/load. Total RNA was extracted from these infected cells and was quantified by real-time polymerase chain reaction (Real-time PCR). The viral titer was compared with the control samples to determine the anti-HCV activity of kaolin derived compounds. Kaolin is a group of clay minerals, with the chemical composition Al 2 Si 2 O 5 (OH) 4 . Results The results showed promising effectiveness of local kaolin derived anti-HCV compounds by causing 28% to 77% decrease in the HCV titer, when applied to infected Huh-7 cell lines. This study provides the basis for future work on these compounds especially to determine the specific pathway and mechanism for inhibitory action in the replicon systems of viral hepatitis. Conclusions Kaolin mineral derivatives show promising inhibitory effects against HCV genotypes 3a and 1a infection, which suggests its possible use as complementary and alternative medicine for HCV viral infection.

Background Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model. Methods We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40 th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells. Results HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40 th day, while HCV core protein was detected from the second day up to 40 th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24 th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3 rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA. Conclusion Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

Background and Aims Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression ( P < 0.05) of EPO was observed in the medium from Huh-7 cell line. Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.