Interaction between LINC-ROR and Stemness State in Gastric Cancer Cells with Helicobacter pylori Infection

Background:LINC-ROR, as a cancer-related lncRNA, has vital roles in stem cell survival, pluripotency, differentiation, and self-renewal in hESCs. However, cancer-related molecular mechanisms, its functional roles, and clinical value of LINC-ROR in GC remain unclear. In this study, we aimed to investigate probable interplay between LINC-ROR with SALL4 stemness regulator and their role with the development of the disease. Methods:The mRNA expression profile of LINC-ROR and SALL4 was assessed in tumoral and adjacent non-cancerous tissues of GC patients, using quantitative real-time PCR. Results:Significant LINC-ROR underexpression and SALL4 overexpression were observed in 55.81% and 75.58% (p < 0.0001) of samples, respectively. The expression of LINC-ROR and SALL4 were significantly correlated with each other (p = 0.044). There was an association between the underexpression of LINC-ROR and sex, stage of tumor progression, tumor type, and location of tumor (p < 0.05), and H. pylori infection with SALL4 expression (p = 0.036). There were also significant correlations between concomitant mRNA expression of SALL4 and LINC-ROR in tumors located at distal noncardiac, positive for H. pylori infection, tumors with invasion into the muscle layer of the stomach, and grade II tumor (p < 0.05). Conclusion:The clinical results of the SALL4-LINC-ROR association propose a probable functional interaction between these markers in tumor maintenance and aggressiveness. Our study can help to understand one of the mechanisms involved in the progression of GC through the function of these regulators. Key Words: LINC-ROR, Non-coding RNA, SALL4     

A Novel Splice Site Variant in the LDLRAP1 Gene Causes Familial Hypercholesterolemia

Background:FH, a hereditary disorder, is caused by pathogenic variants in the LDLR, APOB, and PCSK9 genes. This study has assessed genetic variants in a family, clinically diagnosed with FH. Methods:A family was recruited from MASHAD study in Iran with possible FH based on the Simon Broom criteria. The DNA sample of an affected individual (proband) was analyzed using WES, followed by bioinformatics and segregation analyses. Results:A novel splice site variant (c.345-2A>G) was detected in the LDLRAP1 gene, which was segregated in all affected family members. Moreover, HMGCR rs3846662 g.23092A>G was found to be homozygous (G/G) in the proband, probably leading to reduced response to simvastatin and pravastatin. Conclusion:LDLRAP1 c.345-2A>G could alter the PTB, which acts as an important part of biological pathways related to lipid metabolism. Key Words: Genetic research, LDLRAP1, Hypercholesterolemia, Hydroxymethylglutaryl-CoA Reductase Inhibitors     

Expression Analysis of mir-21 and mir-221 in Cancerous Tissues from Iranian Patients with Gastric Cancer

Background:

Early detection is a key to survival for gastric cancer. Molecular markers such as miRNA (microRNA) can have great importance in the early diagnosis of gastric cancer. Expression of miR-21 and miR-221 are deregulated in many types of human cancers. This study aimed to investigate the differences in miRNA expression patterns within the Iranian population.

Methods:

Total RNA was extracted from gastric cancer tissues and adjacent non-cancerous tissues from 32 patients. Expression levels of miR-21 and miR-221 were detected by Real time RT-PCR using a specific primer, with 5s rRNA as the internal reference gene.

Results:

Our data showed that the expression levels of miR-21 and miR-221 in gastric cancer samples were significantly higher than in paired non-cancerous samples (P < 0.05). The receiver operating characteristic (ROC) analyses yielded the area under the curve (AUC) values of 80.30 for miR-21 and 93.30 for miR-221, and combined ROC analysis revealed the highest AUC value of 96.90 in discriminating GC patients from healthy controls.

Conclusion:

It seems that miR-21 and miR-221 expression pattern in Iranian patients with gastric cancer are similar to any other population. Considering the increased expression level of two miRNAs in cancerous tissue compared to normal tissue as well as the area under ROC curve, miR-21 and miR-221 can be used for early detection of gastric cancer. Key Words: MicroRNAs, Tumor markers, Stomach neoplasms     

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles

Background:hESCs-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods:Herein, hESCs-MSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results:MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. The hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion:Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications. Key Words: Human embryonic stem cells, Mesenchymal stem/stromal cells, Regenerative medicine     

Anti-Proliferative Properties,Biocompatibility, and Chemical Composition of Different Extracts of Plantago major Medicinal Plant

Background:To study the anticancer activity of Plantago major, we assessed the effect of ethanolic, methanolic and acetonic extracts of this plant on HCT-116, SW-480, and HEK-293 cell lines as control. Methods:The cytotoxic activity, biocompatibility, and toxicity were evaluated by MTT assay, hemolysis, and Artemia salina-LD₅₀ (on mice) tests, respectively. The analysis of the extracts was performed by GC-MS analysis. Results:The results showed that all the extracts had the most antiproliferative properties on the HCT-116 cell line. The P. major root extract was more effective than the aerial parts, and IC₅₀ values for ethanolic, methanolic and acetonic root extracts were 405.59, 470.16, and 82.26 µg/mL, respectively on HCT-116 cell line at 72 h. Hemolysis degree of the ethanolic extract of aerial and root parts were approximately 1% at 400 μg/mL.. Using the ethanolic extracts, the Artemia survived every concentration, and no toxicity was observed. One week after the oral administration of different parts of P. major extracts, none of the mice died, even those were administered 2000 mg/kg. The results of GC/MS analysis showed that P. major extracts contain potential anticancer compounds, such as stearic acid (8.61%) in aerial parts of methanolic extract and 1,2- Benzenedicarboxylic acid, mono(2-ethylhexyl)ester (88.07% and 40.63%) in aerial and root parts of acetonic extract of P. major. Conclusions:Our findings suggest that the P. major is a source of potential compounds with antiproliferative properties. Key Words: Gas chromatography-mass spectrometry, HCT-116 cells, Hemolysis, Lethal dose 50     

Cloning,Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium,Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS-PAGE. Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results:Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS- PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion:In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity. Key Words: Escherichia coli, Gene expression, Lactobacillus, Lipase, Phylogeny     

Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus

Background:CE is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods:The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results:Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion:Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice. Key Words: Echinococcus granulosus, Lactococcus lactis, Immunization, Vaccines     

Comparison Study on the Effect of Mesenchymal Stem Cells-Conditioned Medium Derived from Adipose and Wharton’s Jelly on Versican Gene Expression in Hypoxia

Background:Mesenchymal stem cells enhance tissue repair through paracrine effects following transplantation. The versican protein is one of the important factors contributing to this repair mechanism. Using MSC conditioned medium for cultivating monocytes may increase versican protein production and could be a good alternative for transplantation of MSCs. This study investigates the effect of culture medium conditioned by human MSCs on the expression of the versican gene in PBMCs under hypoxia-mimetic and normoxic conditions. Methods:The conditioned media used were derived from either adipose tissue or from WJ. Flow cytometry for surface markers (CD105, CD73, and CD90) was used to confirm MSCs. The PBMCs were isolated and cultured with the culture media of the MSC derived from either the adipose tissue or WJ. Desferrioxamine and cobalt chloride (200 and 300 µM final concentrations, respectively) were added to monocytes media to induce hypoxia-mimetic conditions. Western blotting was applied to detect HIF-1α protein and confirm hypoxia-mimetic conditions in PBMC. Versican gene expression was assessed in PBMC using RT-PCR. Western blotting showed that the expression of HIF-1α in PBMC increased significantly (p < 0.01). Results:RT-PCR results demonstrated that the expression of the versican and VEGF genes in PBMC increased significantly (p < 0.01) in hypoxia-mimetic conditions as compared to normoxia. Conclusion:Based on the findings in the present study, the secreted factors of MSCs can be replaced by direct use of MSCs to improve damaged tissues.Key Words: Adipose tissue, Hypoxia, Mesenchymal stem cells, Wharton’s jelly     

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells

Background:Bispecific antibodies represent an important class of mAbs, with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti- CD3 × CD19 bsAb is a bispecific T-cell engager (BiTE) currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods:WPRE sequence was incorporated at the 3’ end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion:Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in HEK-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody. Key Words: Acute lymphoblastic leukemia, Bispecific antibodies, Monoclonal antibody     

Gloeothece sp.—Exploiting a New Source of Antioxidant,Anti-Inflammatory,and Antitumor Agents

Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from Gloeothece sp.–using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant (ABTS^•+, DPPH^•, ^•NO and O₂^•assays), anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity (death by TUNEL, and anti-proliferative by BrdU incorporation assay in AGS cancer cells); while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge ^-NO^• radical (IC₅₀, 1258 ± 0.353 µg·mL⁻¹); (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL⁻¹; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL⁻¹. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.     

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression

Background:

Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors.

Methods:

In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48, or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P<0.05 was considered statistically significant.

Results:

Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05).

Conclusions:

Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.Key Words: Adiponectin, Adiponectin receptors, Endometriosis, Stromal cells     

Improvement of bacterial blight resistance of hybrid rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon)

A novel bacterial blight (BB) resistance gene, Xa23, identified from Oryza rufipogon was introgressed into three popular restorer lines (Minghui63, YR293 and Y1671) for wild abortive cytoplasmic male sterility by marker-assisted backcross breeding approach in combination with artificial inoculation and stringent phenotypic selections. The three derived BB resistant restorer lines (Minghui63-Xa23, YR293-Xa23 and Y1671-Xa23) and their hybrid combinations with Zhenshan97A (Shanyou63-Xa23), NongfengA (Fengyou293-Xa23) and Zhong9A (Zhongyou1671-Xa23) demonstrated similar BB resistance spectrum as the donor parent, CBB23 (B). The newly developed BB resistant restorers and their derived hybrids were identical to their respective original versions for agronomic traits especially under disease free condition. However, under severe disease condition, the three BB resistant restorer lines exhibited significantly higher grain weight and spikelet fertility as compared to the respective original restorer lines thus further resulting in BB resistant hybrids with significantly higher grain yields than their respective popular original hybrids. The results indicated that the Xa23 gene could completely express its dominant broad spectrum resistance in different backgrounds of both restorer and male sterile lines across different growth stages, suggesting its immense breeding value in BB resistance improvement for hybrid rice. Moreover, a reasonable utilization and deployment of Xa23 gene for efficient control of BB disease in hybrid rice production was recommended.