Serotonin type-3 receptor antagonists selectively kill melanoma cells through classical apoptosis, microtubule depolymerisation, ERK activation, and NF-κB downregulation.

Malignant melanoma is a highly metastatic tumour, resistant to treatment. Serotonin type-3 (5-HT3) receptor antagonists, such as tropisetron and ondansetron, are well-tolerated antiemetic drugs commonly used to prevent nausea caused by chemotherapy or radiotherapy. We investigated the anticancer effects of these drugs on melanoma cancer cell lines WM-266-4 and B16F10 with or without paclitaxel. We constructed IC50 curves and performed Chou-Talalay analysis, using data obtained with the MTT assay. Flow cytometry and fluorescent microscopy were used to examine characteristics of the cell cycle, cell death and cytoskeleton changes. Protein levels and activation were analysed by western blotting and molecular docking studies carried out. Data were analysed by one way ANOVA and post hoc testing. Ondansetron and tropisetron showed selective concentration-dependent cytotoxicity in melanoma cell lines WM-266-4 and B16F10. The effect in combination with paclitaxel was synergistic. The drugs did not cause cell cycle arrest but did promote characteristics of classical apoptosis, including accumulation of subG1 DNA, cleaved caspase-3, mitochondrial membrane permeability and phosphatidylserine exposure. As well, the cytosolic calcium level in the melanoma cells was enhanced, phosphorylated ERK1/2 induced and NF-κB inhibited. Finally, the formation of microtubules was shown to be impaired in melanoma cells treated with ondansetron or tropisetron. Docking studies were used to predict that these drugs could bind to the colchicine binding site on the tubulin molecule. Antiemetic drugs, already given in combination with chemotherapy, may enhance the cytotoxic effect of chemotherapy, following successful delivery to the tumour site.

Employment of Spore-Forming Probiotics to Combat Persister Cells of Staphylococcus Epidermidis.

Background: In this study, spore-forming probiotics were employed to eradicate Staphylococcus epidermidis biofilms and the presence and expression of genes involved in stress response was examined. Methods: Polymerase chain reaction (PCR) assay was used to detect rpoS, relA and mazF genes in S. epidermidis ATCC 12228. Biofilm production was investigated by microtiter plate (MTP) assay. 100X minimum inhibitory concentration (MIC) of gentamycin was used to induce persister cells in planktonic and biofilm bacterial cells. The expression of rpoS, relA, and mazF genes was assessed at different time intervals of 2, 8, and 24 h using real-time PCR assay. Then, dilutions of 1, 0.5, and 0.25 µg/ml of the supernatant of Bacillus coagulans culture was used to eradicate the persister cells and the number of colonies was determined. Results: Persister cells of S. epidermidis were formed after 7 h in planktonic and 5 h in the biofilm structure after exposure to 50 µg/ml of gentamycin. The expression of mazF and rpoS in biofilm structure and the expression of rpoS and relA in persister cells were significantly higher compared to the control (p< 0.05). The number of persister cells showed a reduction of log 2.4 and log 0.8 after exposure to 1 and 0.5 µg/ml B. coagulans supernatant, respectively, but no reduction was observed at the concentration of 0.25 µg/ml. Conclusion: The results showed that the supernatant of probiotics containing their secretive metabolites can be used as a novel approach to combat persister cells.

Methods to Assess Chemokine Binding and Anti-chemotactic Activity of Virus Proteins.

Chemokines are key instigators of inflammatory and immune responses. Viruses can suppress these responses by secreting proteins that interfere with chemokine action. These proteins bind to chemokines and block the host's ability to recruit immune cells to sites of infection, thus facilitating virus replication and spread. When produced recombinantly, chemokine binding proteins provide a formidable resource to deploy against human disease. Here, we describe an enzyme-linked immunosorbent inhibition assay and a chemotaxis inhibition assay that are employed to assess the chemokine binding strength and anti-chemotactic activity of viral proteins. These assays are quick and reproducible, and are thus ideal for screening putative or modified chemokine binding proteins as the first step in their development as therapeutics.

Antiproliferative activity of CD44 siRNA-PEI-PEG nanoparticles in glioblastoma: involvement of AKT signaling.

BACKGROUND AND PURPOSE: Glioblastoma multiforme (GBM) is the most invasive type of cancer which starts inside the brain. GBM cells were found to have similar properties to glioblastoma cancer stem cells. CD44 can be used as a marker of the cancer stem cells in a subset of glioblastoma tumor cells. Recent studies showed that CD44 is involved in developing cancer cells via the protein kinase B (PKB or AKT) signaling pathway. Therefore, this study aimed to investigate the CD44 mRNA silencing effects on the glioblastoma cell cycle via AKT signaling pathway. EXPERIMENTAL APPROACH: To determine CD44 expression in the samples of the patients with GBM, we used the analysis of data extracted from TCGA database. qRT-PCR and western blotting were used to evaluate the expression level of genes and proteins. Different cell cycles were evaluated by DAPI staining and flow cytometry. FINDINGS/RESULTS: Bioinformatics results showed that CD44 expression level in GBM tumor samples is higher than in normal samples. Effects of poly (ethylene imine)-polyethylene glycol (PEI-PEG)-loaded CD44 siRNA in cell cycle showed that CD44 silencing could inhibit cell cycle in G0-G1 phase by more than 20% compared to the negative control (P < 0.05). Furthermore, PEI-PEG-loaded CD44 siRNA reduces the expression of cyclin D1 and CKD-4. According to our findings, this structure also prevented AKT phosphorylation at Thr-308 and Ser-473. CONCLUSION AND IMPLICATIONS: Our results suggest that PEI-PEG-loaded CD44 siRNA may attenuate the cell cycle by suppressing AKT signaling pathway.

Association of CATSPER1, SPATA16 and TEX11 genes polymorphism with idiopathic azoospermia and oligospermia risk in Iranian population.

BACKGROUND: Male infertility is a heterogeneous disease which can occur due to spermatogenesis defects. The idiopathic azoospermia and oligospermia are the common cause of male infertility with unknown underlying molecular mechanisms. The aim of this study was to investigate association of idiopathic azoospermia and oligospermia with single-nucleotide polymorphisms of CATSPER1, SPATA16 and TEX11 genes in Iranian-Azeri men. METHODS: In this case-control study, we recruited 100 infertile men (case group) and 100 fertile men (control group) from Azeri population in north western provinces, Iran, population. The genomic DNA was extracted using a proteinase K method from peripheral blood leukocytes. The genotypes analysis was conducted using tetra-primer amplification refractory mutation system-polymerase chain reaction method. The obtained data were analyzed by statistical software. RESULTS: We found a significant difference in the frequencies of heterozygote AB and mutant homozygote BB genotypes in the CATSPER1 (rs2845570) gene polymorphism between patients and healthy controls (p < 0.05). Moreover, we observed a significant difference in the frequencies of heterozygote BA genotype in the SPATA16 (rs1515442) gene polymorphism between patients and healthy controls (p < 0.05). However, no significant difference was found in genotypes distribution of case and control groups in the TEX11 (rs143246552) gene polymorphism. CONCLUSION: Our finding showed that the CATSPER1 (rs2845570) and SPATA16 (rs1515442) genes polymorphism may play an important role in idiopathic azoospermia and oligospermia in Iranian Azeri population. However, more extensive studies with larger sample sizes from different ethnic origins are essential for access more accurate results.

The Immune Response to SARS-CoV-2 and Variants of Concern.

At the end of 2019 a newly emerged betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of an outbreak of severe pneumonia, subsequently termed COVID-19, in a number of patients in Wuhan, China. Subsequently, SARS-CoV-2 rapidly spread globally, resulting in a pandemic that has to date infected over 200 million individuals and resulted in more than 4.3 million deaths. While SARS-CoV-2 results in severe disease in 13.8%, with increasing frequency of severe disease with age, over 80% of infections are asymptomatic or mild. The immune response is an important determinant of outcome following SARS-CoV-2 infection. While B cell and T cell responses are associated with control of infection and protection against subsequent challenge with SARS-CoV-2, failure to control viral replication and the resulting hyperinflammation are associated with severe COVID-19. Towards the end of 2020, several variants of concern emerged that demonstrate increased transmissibility and/or evasion of immune responses from prior SARS-CoV-2 infection. This article reviews what is known about the humoral and cellular immune responses to SARS-CoV-2 and how mutation and structural/functional changes in the emerging variants of concern impact upon the immune protection from prior infection or vaccination.

Distal Renal Tubular Acidosis in an Iranian Patient with Hereditary Spherocytosis

Background: Hereditary spherocytosis (HS) and hereditary hereditary distal renal tubular acidosis (dRTA) are associated with mutations in the SLC4A1 gene encoding the anion exchanger 1. In this study, some patients with clinical evidence of congenital HS and renal symptoms were investigated. Methods: Twelve patients with congenital HS and renal symptoms were recruited from Ali-Asghar Children's Hospital (Tehran, Iran). A patient suspected of having dRTA was examined using whole exome sequencing method, followed by Sanger sequencing. Results: One patient (HS03) showed severe failure to thrive, short stature, frequent urinary infection, and weakness. A homozygote (rs571376371 for c.2494C>T; p.Arg832Cys) and a heterozygote (rs377051298 for c.466C>T; p.Arg156Trp) missense variant were identified in the SLC4A1 and SPTA1 genes, respectively. The compound heterozygous mutations manifested as idRTA and severe HS in patient HS03. Conclusion: Our observations, for the first time, revealed clinical and genetic characteristics of idRTA and severe HS in an Iranian patient HS03.

Inhibitory Effect of Bismuth Oxide Nanoparticles Produced by Bacillus licheniformis on Methicillin-Resistant Staphylococcus aureus Strains (MRSA).

BACKGROUND: Based on the increase in antibiotic-resistant pathogens, it is necessary to have various effective compounds, so as to prevent its proliferation of these pathogens. For this purpose, nano-materials such as bismuth oxide nanoparticles can be used. OBJECTIVES: The aim of this study was to produce bismuth oxide nanoparticles by Bacillus licheniformis PTCC1320 and to determine the antimicrobial effects on methicillin-resistant Staphylococcus aureus species compared with some antibiotics. MATERIALS AND METHODS: In this study, 200 bacterial samples were collected from hospitalized patients with burn infections from the Burn Rescue Hospital, Tehran. Thereafter, 65 strains of methicillin-resistant Staphylococcus aureus were identified by their phenotype and genotype. A total of 92% of identified strains with the highest resistance to antibiotics were isolated. Bismuth oxide nanoparticles were synthesized by Bacillus licheniformis PTCC1320. FTIR spectroscopy, X-ray diffraction, and scanning electron microscopy (SEM) were used to analyze the extracellularly produced nanoparticles. Finally, the antibacterial properties of nanoparticles produced on the biofilm of some pathogens were examined. RESULTS: In the present study, cube-shaped bismuth oxide nanoparticles were formed in the size range of 29-62 nm. They were found to have antimicrobial activity on 16% of the isolated Staphylococcus aureus strains. The FTIR results showed the vibrational frequencies of bismuth oxide at 583, 680, 737, and 1630 nm. The XRD results also confirmed the structure of nanoparticles. Compared with antibiotics such as Ciprofloxacin, bismuth oxide nanoparticles had less affectivity on this resistant hospital pathogen. Increasing the concentration of bismuth oxide nanoparticles, increased its antimicrobial effect and decreased bacterial growth rate. CONCLUSION: Compared with heavy metals, bismuth nanoparticles have very low antibacterial effects. Considering this feature, the use of less antibiotics can be achieved with bismuth nanoparticles in the treatment of infections, thereby reducing antibiotic resistance.

Recombinant HCV core protein and the secretion of associated cytokines (IL-6, TNF-α and IFN-γ) in immunized mice.

Hepatitis C virus (HCV) is an important cause of acute and chronic hepatitis which is a disorder with a high worldwide prevalence. HCV core protein was considered as immunogenic counterpart of the HCV vaccine and it is an ideal candidate for HCV vaccine. Since cytokines such as IL-6, TNF-alpha and IFN-Gamma are responsible for the prevention of viral infection, this study aimed to evaluate the effectiveness of HCV core protein as a vaccine. Ten BALB/c mice were immunized with HCV core protein and after 42 days the splenocytes were isolated and the IL-6 and INF-gamma secretion were measured using ELISpot technique, at the same time TNF-alpha was determined by ELISA in the sera. The MTT assay was done to assess the viability of the cultured splenocytes. For evaluating the humoral immune response against the recombinant HCV core protein the DOT Blot test was used. Data was compared using one-way ANOVA test and significant results were considered at p < 0.05. In the present study the IL-6, INF-gamma and TNF-alpha levels were dramatically higher in the immunized mice compared to the control group (respectively, 22.9 +/- 1.26; 18.53 +/- 3.87; 53.96 +/- 4.54 and p < 0.05). The immunized mice with recombinant HCV core protein showed higher amount of IL-6, INF-gamma and TNF-alpha in the current study. Since the level of IL-6, TNF-alpha and IFN-gamma is high in patients with acute HCV infection, thus a vaccine which could stimulate the secretion of these cytokines in advance may have a preventive role.

The evaluation of hepatitis C virus core antigen in immunized BALB/c mice.

BACKGROUND: Hepatitis infection represents one of the important causes of morbidity and mortality in developing countries, however there is not any effective vaccine against hepatitis C which is one of the significant problems in vaccine project. OBJECTIVES: The aim of the present study is to evaluate the role of HCV core protein in inducing IFN-Gamma secretion and TCL activities as a vaccine in Balb/C mice. MATERIAL AND METHODS: Our previous cloned plasmid (HCV Core gene into pETDuet-1) applied for protein expression in bacteria. The expressed and purified recombinant protein together with Freund's adjuvant was injected to 15 Balb/c mice. The total IgG and IgG2a of immunized mice sera were evaluated after a week. Two weeks after booster injection, we studied the proliferation and IFNγ secretion of spleens, inguinal and popliteal lymph nodes lymphocytes by ELISA and ELISPOT. RESULTS: The FSFC (Frequency of spot forming cells) of secreting cells of immunized mice with HCV/Core protein and sera IgG2a were considerably higher than the control groups. CONCLUSIONS: The core protein together with proper adjuvant can be a candidate vaccine against of HCV infection.