The Safe Soluble Compound Dehydroascorbic Acid Inhibits Various Upstream and Downstream Effectors of PI3K and KRAS Signaling Pathways in Undruggable PIK3CA/KRAS-Mutant Colorectal Cancer Stem-Like Cells.

Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS-mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS-mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA/KRAS-mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 µM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4, Bmi-1, Sox-2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 µM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro. L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4, stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS-mutant CRCSCs using L-DHA with no toxicity on normal cells.

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human ß-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity - an integrated experimental and bioinformatic approach.

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence.Communicated by Ramaswamy H. Sarma.

Phytochemical constituents and biological activities of Salvia macrosiphon Boiss.

Salvia macrosiphon Boiss. is an aromatic perennial herb belonging to the family Lamiaceae. Phytochemical studies and biological activities of this plant have been rarely documented in the literature. The current study aimed to investigate antibacterial and cytotoxic activity of different fractions of aerial parts of S. macrosiphon. Also, we tried to isolate and identify cytotoxic compounds from the plant. In this respect, the hydroalcoholic extract of the corresponding parts of the plant was fractionated into four fractions. Then, antibacterial and cytotoxic activity of each fraction were examined. It was found that the chloroform fraction had a good antibacterial activity against gram-positive and gram-negative bacteria. The most potent cytotoxicity was also obtained by the n-hexane fraction comparing with etoposide as the reference drug which was selected for the study and characterization of secondary metabolites. Accordingly, 13-epi manoyl oxide (1), 6α-hydroxy-13-epimanoyl oxide (2), 5-hydroxy-7,4'-dimethoxyflavone (3), and ß-sitosterol (4) were isolated and evaluated for their cytotoxic activity. Among them, compound 1 revealed significant cytotoxicity against A549, MCF-7, and MDA-MB-231. It merits mentioning that it showed high selectivity index ratio regarding the low cytotoxic effects on Human Dermal Fibroblast which can be considered as a promising anticancer candidate.

Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells.

BACKGROUND: Human FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3'-end formation of mRNAs are required. OBJECTIVES: We aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells. MATERIALS AND METHODS: HepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells. RESULTS: Our data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs. CONCLUSIONS: The embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.

Effective Targeting Survivin, Caspase-3 and MicroRNA-16-1 Expression by Methyl-3-pentyl-6-methoxyprodigiosene Triggers Apoptosis in Colorectal Cancer Stem-Like Cells.

Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential biomarkers could be targeted in CRC initiating cells with stem-like cells properties by prodigiosin and this compound with high pro-apoptotic capacity represents the possibility of its therapeutic application directed against CCSCs.

Estimation the Frequency of Human Immunodeficiency Virus among Male and Female Patients, Iran.

OBJECTIVE: A reduction in new human immunodeficiency virus(HIV) cases is one of the ten areas prioritized by the United Nations Program on HIV. However, recent official reports confirm the HIV rate is increasing and predicted a huge incidence in the near future in Iran, despite the preventative program by Iran's Health Ministry. In this descriptive study, we evaluate the frequency of HIV positive cases among referral patients to a private clinic laboratory for its diagnosis in addition to specimens from other laboratories. An epidemiological analysis is also performed. MATERIALS AND METHODS: In this descriptive study, the total number of patients was 138 cases that referred for the diagnosis of HIV to the private Laboratory. Of these, 93 males (67.4%) and 45 females (32.6%) voluntarily requested to be examined for specific increases in specific antibody titer, western blot assays and RNA quantitation polymerase chain reaction. We collected two separate tubes of whole blood, one for reverse transcriptasepolymerase chain reaction analysis and the second one for the remaining two tests. Those patients who were antibody positive by western blot and/or reverse transcriptase-polymerase chain reaction(RT-PCR) analyses were considered as HIV positive cases. RESULTS: There were 18.84% confirmed HIVcases (17.39% males; 1.45% females). Analysis of the results confirmed that the ratio of male to female patients in the infected group was not comparable to those in the suspect group. The majority of HIV positive cases were either infected by their partner via sexual intercourse (84.61%) or needle sticks (11.53%) among the drug addicted group. The infection routes of the remainder were unknown. CONCLUSION: Analysis of the data revealed a higher frequency of HIVin males than females among the tested group. There was a shift in to unsafe sexual intercourse as seen in the present study. The higher rate of infected male patients shows a shift in transmission route to unsafe intercourse. Therefore, it is necessary to design new supportive programs by actively identifying and contacting at-risk groups, particularly infected females who are uninterested in being and monitored.