One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers. Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR. Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR. Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories.

Circulating Tumor Cells as a Promising Tool for Early Detection of Hepatocellular Carcinoma.

Liver cancer is a significant contributor to the cancer burden, and its incidence rates have recently increased in almost all countries. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is the second leading cause of cancer-related deaths worldwide. Because of the late diagnosis and lack of efficient therapeutic modality for advanced stages of HCC, the death rate continues to increase by ~2-3% per year. Circulating tumor cells (CTCs) are promising tools for early diagnosis, precise prognosis, and follow-up of therapeutic responses. They can be considered to be an innovative biomarker for the early detection of tumors and targeted molecular therapy. In this review, we briefly discuss the novel materials and technologies applied for the practical isolation and detection of CTCs in HCC. Also, the clinical value of CTC detection in HCC is highlighted.

Combined Effect of Neutron Radiation and Curcumin on Breast Cancer Cells Cytotoxicity in 3D Culture Medium

Introduction: Introduction: Chemotherapy, biotherapy, and radiotherapy play a limited but important role in treating breast cancer. For more efficient treatment, combination therapy could be an appropriate option. In this study, radiotherapy using neutron radiation emitted from a 241Am-Be neutron source, as well as biotherapy using curcumin (80 µM) was combined to investigate the efficiency of treatment towards MCF-7 breast cancer in a three dimensional (3D) culture medium. Methods: Methods: MTT, neutral red uptake assay, nitric oxide, glutathione assay, catalase, cytochrome c, comet assay, and caspase-3 were used to determine the effect of neutron radiation and also neutron and curcumin combination on the viability of cancer cells. Results: Results: The results of cytotoxicity test showed that neutron irradiation with or without curcumin at 5, 10, 15, and 20 h reduced the survival of tumor cells. Moreover, the rate of apoptosis due to the neutron effect at different irradiation times enhanced with the increasing time. Conclusion: Conclusion: Due to the significant anticancer effect of curcumin in 3D culture, using this molecule before or after neutron therapy is recommended.

Evaluation of Anticancer and Cytotoxic Effects of Genistein on PC3 Prostate Cell Line under Three-Dimensional Culture Medium

Background: Prostate cancer is a major cause of disease and mortality among men. Genistein (GNT) is an isoflavone found naturally in legumes. Isoflavones, a subset of phytoestrogens, are structurally similar to mammalian estrogens. This study aimed to evaluate the anticancer and cytotoxic effects of GNT on PC3 cell line under three dimensional (3D) culture medium. Methods: The 3D culture was created by encapsulating the PC3 cells in alginate hydrogel. MTT assay, neutral red uptake, comet assay, and cytochrome C assay were used to study the anticancer and cytotoxic effects of GNT at 120, 240, and 480 µM concentrations. Also, nitric oxide (NO), catalase, and glutathione assay levels were determined to evaluate the effect of GNT on the cellular stress. The culture medium was used as the negative control. Results: GNT reduced the production of cellular NO and increased the production of catalase and glutathione, confirming the results of the NO test. Evaluation of the toxicity effect of GNT at the concentrations of 120, 240, and 480 µM using comet assay showed that this chemical agent induces apoptosis in PC3 cells in a dose-dependent manner. As the level of cytochrome C in PC3 cells treated with different concentrations of GNT was not significantly different from that of the control, GNT could induce apoptosis in PC3 cells through the non-mitochondrial pathway. Conclusion: The findings of this study disclose that the anticancer effect of GNT on PC3 cells under 3D culture conditions could increase the effectiveness of treatment. Also, the cell survival rate is dependent on GNT concentration.

Novel Gene-Correction-Based Therapeutic Modalities for Monogenic Liver Disorders.

The majority of monogenic liver diseases are autosomal recessive disorders, with few being sex-related or co-dominant. Although orthotopic liver transplantation (LT) is currently the sole therapeutic option for end-stage patients, such an invasive surgical approach is severely restricted by the lack of donors and post-transplant complications, mainly associated with life-long immunosuppressive regimens. Therefore, the last decade has witnessed efforts for innovative cellular or gene-based therapeutic strategies. Gene therapy is a promising approach for treatment of many hereditary disorders, such as monogenic inborn errors. The liver is an organ characterized by unique features, making it an attractive target for in vivo and ex vivo gene transfer. The current genetic approaches for hereditary liver diseases are mediated by viral or non-viral vectors, with promising results generated by gene-editing tools, such as CRISPR-Cas9 technology. Despite massive progress in experimental gene-correction technologies, limitations in validated approaches for monogenic liver disorders have encouraged researchers to refine promising gene therapy protocols. Herein, we highlighted the most common monogenetic liver disorders, followed by proposed genetic engineering approaches, offered as promising therapeutic modalities.

Regulatory Non-Coding RNAs in Familial Hypercholesterolemia, Theranostic Applications.

Familial hypercholesterolemia (FH) is a common monogenic disease which is associated with high serum levels of low-density lipoprotein cholesterol (LDL-C) and leads to atherosclerosis and cardiovascular disease (CVD). Early diagnosis and effective treatment strategy can significantly improve prognosis. Recently, non-coding RNAs (ncRNAs) have emerged as novel biomarkers for the diagnosis and innovative targets for therapeutics. Non-coding RNAs have essential roles in the regulation of LDL-C homeostasis, suggesting that manipulation and regulating ncRNAs could be a promising theranostic approach to ameliorate clinical complications of FH, particularly cardiovascular disease. In this review, we briefly discussed the mechanisms and pathophysiology of FH and novel therapeutic strategies for the treatment of FH. Moreover, the theranostic effects of different non-coding RNAs for the treatment and diagnosis of FH were highlighted. Finally, the advantages and disadvantages of ncRNA-based therapies vs. conventional therapies were discussed.

Rhenium Perrhenate (188ReO4) Induced Apoptosis and Reduced Cancerous Phenotype in Liver Cancer Cells.

Recurrence in hepatocellular carcinoma (HCC) after conventional treatments is a crucial challenge. Despite the promising progress in advanced targeted therapies, HCC is the fourth leading cause of cancer death worldwide. Radionuclide therapy can potentially be a practical targeted approach to address this concern. Rhenium-188 (188Re) is a ß-emitting radionuclide used in the clinic to induce apoptosis and inhibit cell proliferation. Although adherent cell cultures are efficient and reliable, appropriate cell-cell and cell-extracellular matrix (ECM) contact is still lacking. Thus, we herein aimed to assess 188Re as a potential therapeutic component for HCC in 2D and 3D models. The death rate in treated Huh7 and HepG2 lines was significantly higher than in untreated control groups using viability assay. After treatment with 188ReO4, Annexin/PI data indicated considerable apoptosis induction in HepG2 cells after 48 h but not Huh7 cells. Quantitative RT-PCR and western blotting data also showed increased apoptosis in response to 188ReO4 treatment. In Huh7 cells, exposure to an effective dose of 188ReO4 led to cell cycle arrest in the G2 phase. Moreover, colony formation assay confirmed post-exposure growth suppression in Huh7 and HepG2 cells. Then, the immunostaining displayed proliferation inhibition in the 188ReO4-treated cells on 3D scaffolds of liver ECM. The PI3-AKT signaling pathway was activated in 3D culture but not in 2D culture. In nude mice, Huh7 cells treated with an effective dose of 188ReO4 lost their tumor formation ability compared to the control group. These findings suggest that 188ReO4 can be a potential new therapeutic agent against HCC through induction of apoptosis and cell cycle arrest and inhibition of tumor formation. This approach can be effectively combined with antibodies and peptides for more selective and personalized therapy.

Application of Stem Cell-Derived Extracellular Vesicles as an Innovative Theranostics in Microbial Diseases.

Extracellular vesicles (EVs), as nano-/micro-scale vehicles, are membranous particles containing various cargoes including peptides, proteins, different types of RNAs and other nucleic acids, and lipids. These vesicles are produced by all cell types, in which stem cells are a potent source for them. Stem cell-derived EVs could be promising platforms for treatment of infectious diseases and early diagnosis. Infectious diseases are responsible for more than 11 million deaths annually. Highly transmissible nature of some microbes, such as newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), drives researcher's interest to set up different strategies to develop novel therapeutic strategies. Recently, EVs-based diagnostic and therapeutic approaches have been launched and gaining momentum very fast. The efficiency of stem cell-derived EVs on treatment of clinical complications of different viruses and bacteria, such as SARS-CoV-2, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Staphylococcus aureus, Escherichia coli has been demonstrated. On the other hand, microbial pathogens are able to incorporate their components into their EVs. The microbe-derived EVs have different physiological and pathological impacts on the other organisms. In this review, we briefly discussed biogenesis and the fate of EVs. Then, EV-based therapy was described and recent developments in understanding the potential application of stem cell-derived EVs on pathogenic microorganisms were recapitulated. Furthermore, the mechanisms by which EVs were exploited to fight against infectious diseases were highlighted. Finally, the deriver challenges in translation of stem cell-derived EVs into the clinical arena were explored.

In-vitro Study of Hottentotta Schach Crude Venom Anticancer Effects on MCF-7 and Vero Cell Lines.

Scorpion venoms contain potentially useful pharmacological agents. Several studies demonstrate that the venoms of some scorpions induce apoptosis and inhibit the growth of cancer cells; therefore, they have been investigated for isolating anticancer components. In this study, antitumor effects of Hottentotta schach crude venom on MCF-7 (breast cancer cell line) as test group and Vero (African green monkey kidney normal cell line) as control group were analyzed. Cell toxicity was analyzed using MTT and neutral red (NR) uptake assays and apoptosis induction was analyzed using comet assay and caspase-3 activity. Oxidative stress following Hottentotta schach crude venom treatment was analyzed using nitrite oxide (NO) determination assay, reduced glutathione (GSH) and catalase enzyme activity assays. Results showed that crude venom (25-200 µg/mL) induced apoptosis and inhibited the growth of MCF-7 and to a lesser extent in Vero cell lines. Nitrite oxide concentration increased while glutathione concentration and catalase enzyme activity were decreased in MCF-7 cells; however, results in Vero cells were reversed completely. It can be concluded that Hottentotta schach crude venom disturbs the oxidation and reduction potential in cancer cells and ultimately induce apoptosis. So this venom can be used as a good source for isolation and designing new anticancer drugs.