Enzymatic and antibacterial activity of the recombinant endolysin PVP-SE1gp146 expressed in Hansenula polymorpha.

Antibiotic resistance, a major global concern, highlights the need for discovering alternative therapies. Recently, endolysins have garnered attention as antibacterial tools with a lower resistance development rate compared to conventional antibiotics, and their production in various expression hosts holds significance. Given its generally recognized as safe (GRAS) status and other advantages, Hansenula polymorpha offers a promising host for endolysin production. PVP-SE1gp146 originates from the Salmonella Enteritidis-specific phage PVP-SE1, which has been previously characterized. We inserted the PVP-SE1gp146 coding gene into the H. polymorpha expression vector pHIPX4. The resulting recombinant, pHIPX4-PVP-SE1gp146, was then introduced into H. polymorpha NCYC495 to facilitate the production of the endolysin PVP-SE1gp146. The expression level of the PVP-SE1gp146 protein was assessed, and it was determined to be approximately 43 mg/l of yeast culture medium. The enzymatic (muralytic) activity of this endolysin was also evaluated, corresponding to the version produced by the E. coli Bl21 strain. The endolysin exhibited admissible antibacterial activity against several gram-negative species, including P. aeruginosa, E. coli, and A. baumannii, while showing an almost negligible impact on K. pneumoniae. Endolysin production within GRAS-approved hosts holds potential for combating antibiotic-resistant bacteria. Challenges involve optimizing concentrations, targeting gram-negative species and improving attachment to bacterial cell walls. Addressing these issues requires dedicated research in endolysin engineering and a comprehensive evaluation of their production in diverse expression hosts.

RNA-sequencing for transcriptional profiling of whole blood in early stage and metastatic pancreatic cancer patients.

We investigated the transcriptional profile of whole blood in early and metastatic stages of pancreatic cancer (PaC) patients to identify potential diagnostic factors for early diagnosis. Blood samples from 18 participants (6 healthy individuals, 6 patients in early stage (I/II) PaC, and 6 patients in metastatic PaC) were analyzed by RNA-sequencing. The expression levels of identified genes were subsequently compared with their expression in pancreatic tumor tissues based on TCGA data reported in UALCAN and GEPIA2 databases. Overall, 331 and 724 genes were identified as differentially expressed genes in early and metastatic stages, respectively. Of these, 146 genes were shared by early and metastatic stages. Upregulation of PTCD3 and UBA52 genes and downregulation of A2M and ARID1B genes in PaC patients were observed from early stage to metastasis. TCGA database showed increasing trend in expression levels of these genes from stage I to IV in pancreatic tumor tissue. Finally, we found that low expression of PTCD3, A2M, and ARID1B genes and high expression of UBA52 gene were positively correlated with PaC patients survival. We identified a four-gene set (PTCD3, UBA52, A2M, and ARID1B) expressed in peripheral blood of early stage and metastatic PaC patients that may be useful for PaC early diagnosis.

Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.

Ectopic expression of insulin in a type 1 diabetic rat model by injection of manipulated mesenchymal stem cells with an insulin construct driven by a glucose-sensitive promoter in the port vein.

The treatment of type 1 diabetes through islet cell transplantation is a complex process, facing challenges such as allograft rejections and a limited supply of donors. One potential solution is to utilize the liver as an alternative for natural insulin production, as hepatocytes can secrete proteins and respond to glucose levels. Recent research has shown promising results in using mesenchymal stem cells as a potential cure for diabetes. The study utilized a diabetic rat model, confirmed through blood sugar measurement. A plasmid vector was designed with specific genetic components, synthesized by biotech company, and then Inserted vector into a plasmid with resistance genes and bacterial origin. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured and transfected with the plasmid using Lipofectamine 3000. Polymerase chain reaction was employed to confirm successful transfection using specific primers. For the animal study, 30 male Wistar rats were divided into six groups, each comprising five rats. The control group did not receive any treatment, while the second group received MSCs via Portal Vein Injection. The third group received MSCs transfected with a specific construct via Portal Vein Injection. The fourth group was induced to develop diabetes through streptozotocin (STZ) injection, the fifth group developed diabetes and received untransfected MSCs via Portal Vein Injection, and the sixth group received MSCs transfected with the specific construct via Portal Vein Injection. To manage Pain, appropriate pain control was administered to the rats for 3 days after the surgery. Fixed liver tissues obtained from the euthanized rats were utilized for immunohistochemistry. In this study, immunohistochemical techniques were used to examine insulin expression in different groups of rats. The control groups showed high levels of insulin expression, while the diabetic groups exhibited lower expression. However, there was a significant difference between the diabetic groups treated with MSC and transgenic MSC cells. All groups had similar baseline glucose levels, but the diabetic groups showed a significant increase after STZ injection, whereas the control and MSC groups did not. Postintervention, both the control and MSC groups had similar glucose levels to the post-STZ levels. However, diabetes-induced groups experienced a significant decrease in glucose levels, with the transfected MSCs showing a greater decrease than the untransfected MSCs. The study suggested that treatment with MSCs, especially transfected ones, can effectively reduce glucose levels in rats with diabetes. In this research, rat BM-MSCs were utilized to create insulin-producing mesenchymal cells with glucose-sensitive insulin expression. The cells were transferred to the liver of diabetic rats via portal vein injection, leading to an increase in insulin expression. This study proposes a novel approach for cell therapy and delivery in the treatment of type 1 diabetes.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.

The OmpA of commensal Escherichia coli of CRC patients affects apoptosis of the HCT116 colon cancer cell line.

BACKGROUND: Colorectal cancer ranks third globally among all types of cancers. Dysbiosis of the gut microbiota of people with CRC is one of the effective agents in the tumorigenesis and metastasis in this type of cancer. The population of Escherichia coli strains, a component of gut microbiota, is increased in the gut of people with CRC compared with healthy people. So, E.coli strains isolated from these patients may have a role in tumorigenesis. Because the most isolated strains belong to the B2 phylogenuetic group, there seems to be a linkage between the bacterium components and malignancy. MATERIAL AND METHODS: In this study, the proteomic comparison between isolated Ecoli from CRC patients and healthy people was assayed. The isolated spot was studied by Two-dimensional gel electrophoresis (2DE) and Liquid chromatography-mass spectrometry (LC-MS). The results showed that the expression of Outer membrane protein A (OmpA) protein increased in the commensal E.coli B2 phylogenetic group isolated from CRC patients. Additionally, we analyzed the effect of the OmpA protein on the expression of the four genes related to apoptosis in the HCT116 colon cancer cell line. RESULTS: This study identified that OmpA protein was overexpressed in the commensal E.coli B2 phylogenetic group isolated from CRC patients compared to the E.coli from the control group. This protein significantly decreased the expression of Bax and Bak, pro-apoptotic genes, as well as the expression of P53 in the HCT116 Cell Line, P < 0.0001. LC-MS and protein bioinformatics results confirmed that this protein is outer membrane protein A, which can bind to nucleic acid and some of the organelle proteins on the eukaryotic cell surface. CONCLUSIONS: According to our invitro and insilico investigations, OmpA of gut E.coli strains that belong to the B2 phylogenetic group can affect the eukaryotic cell cycle.

Utility of blood as the clinical specimen for the diagnosis of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification and real-time polymerase chain reaction assays based on REP-529 sequence and B1 gene.

BACKGROUND: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. METHODS: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. RESULTS: Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. CONCLUSIONS: Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Extracellular vesicles from serum samples of mycobacteria patients induced cell death of THP-1 monocyte and PBMC.

BACKGROUND: Extracellular vesicles (EVs) play a key role in cell communication and the pathogenesis of some diseases. EVs may accelerate cell death during the course of mycobacterial infection and are also considered as a new vaccine design, drug delivery, and biomarker candidates. The current study evaluates the effects of EVs from serum samples of mycobacteria-infected patients on THP-1 monocytes and PBMC cells. METHOD: EVs were purified from the serum, then cultured separately with THP-1 monocytes and PBMCs. The cell death was determined through annexin V-FITC and PI staining. GW4869, an EVs inhibitor, was used to determine if EVs released from serum could increase THP-1 monocytes cell death. RESULTS: The cell death was significantly increased in the presence of 10 µg/ml and 5 µg/ml concentrations of the purified EVs (p < 0.05). Minimal cell death was determined in 2.5 µg/ml and 1.2 µg/ml (p < 0.05). Up to 85% of the cells were viable in the presence of the GW4869 inhibitor (p < 0.05). CONCLUSION: Direct infection of the cells with EVs released from mycobacteria-infected patients samples, the multiplicity of infection with the EVs, and virulent or avirulent mycobacteria may change the status of the cell death. The isolated EVs  from serum samples of patients with mycobacterial  infection accelerated cell death, which means that they might   not be considered as an optimal tool for developing drug delivery and vaccine against tuberculosis.

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.

The utility of dermal fibroblasts in treatment of skin disorders: A paradigm of recessive dystrophic epidermolysis bullosa.

Dermal fibroblasts are the most accessible cells in the skin that have gained significant attention in cell therapy. Applying dermal fibroblasts' regenerative capacity can introduce new patterns to develop cell-based therapies to treat skin disorders. Dermal fibroblasts originate from mesenchymal cells and are located within the dermis. These cells are mainly responsible for synthesizing glycosaminoglycans, collagens, and components of extracellular matrix supporting skin's structural integrity. Preclinical studies suggested that allogeneic and autologous dermal fibroblasts provide widespread and beneficial applications for wound healing, burn ulcers, and inherited skin disorders. In this regard, generating induced pluripotent stem cells (iPSCs) from fibroblasts and gene-edited fibroblasts are promising approaches for treating skin disorders. Here, we aimed to review literature about ongoing and completed clinical trials that applied fibroblasts and bioengineered fibroblasts as therapeutic agents for various skin disorders. This review explores cell therapy protocols from the earliest phase of allogeneic and autologous fibroblasts development in different benches to translating them into bedside-level treatment for skin disorders, particularly recessive dystrophic epidermolysis bullosa.

Halophilic Prokaryotes in Urmia Salt Lake, a Hypersaline Environment in Iran.

In this study, fluorescence in situ hybridization (FISH) and PCR-amplified fragments of the 16SrDNA gene were used to determine prokaryotes diversity in Urmia Salt Lake. Prokaryote cell population in Urmia lake range from 3.1 ± 0.3 × 106, 2 ± 0.2 × 108, 4 ± 0.3 × 108, and 1.8 ± 0.2 × 108 cells ml-1 for water, soil, sediment, and salt samples by DAPI (4́, 6-diamidino-2-phenylindole) direct count, respectively. The proportion of bacteria and archaea in the samples determinable by FISH ranged between 36.1 and 55% and 48.5 and 55.5%, respectively. According to the DGGE method, some bands were selected and separated from the gel, then amplified and sequenced. The results of sequences were related to two phyla Proteobacteria (16.6%) and Bacteroidetes (83.3%), which belonged to four genera Salinibacter, Mangroviflexus, Pseudomonas, and Cesiribacter, and the archaeal sequences were related to Euryarchaeota phyla and three genera Halonotius, Haloquadratum, and Halorubrum. According to our results, it seems that prokaryotic populations in this hypersaline environment are more diverse than expected, and bacteria are so abundant and diverse and form the metabolically active part of the microbial population inhabiting this extreme environment. Molecular dependent and independent approaches revealed a different aspect of this environment microbiota.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

The potential therapeutic role of PTR1 gene in non-healing anthroponotic cutaneous leishmaniasis due to Leishmania tropica.

BACKGROUND: Drug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1. METHODS: Two non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages. RESULTS: All three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05). CONCLUSION: The results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug.

Evaluation of a new multi-epitope sequence of eight known Leishmania infantum antigens for HVL diagnosis by ELISA and Western blot.

BACKGROUND & OBJECTIVES: Leishmaniasis, known as a disease with high prevalence proportion throughout the world, is caused by protozoan parasites. Visceral leishmaniasis is the most severe form of this condition reported sporadically from all regions in Iran. Between different diagnostic tests, serodiagnosis of this infection is of utmost importance in both humans and dogs. Although rK39 ELISA test has been extensively validated in endemic areas, there are currently challenges regarding a more appropriate serodiagnostic test. METHODS: A novel multi-epitope construct was designed consisting of highexposedB cell epitopes using eight important antigens of Leishmania infantum (Gp63, KMP-11, HSP70, CPA, H2A, H3, LACK and TRYP). Our artificial sequence, a Multi-epitope Recombinant Protein (MRP), was consequently produced and purified. Then, immunoreactivity was investigated by ELISA test and western blotting as well. RESULTS: In the present study, the cutoff value (1.052) for the new MRP-ELISA was determined by receiver operator characteristic (ROC) curve analysis using 35 known positive and 20 known negative HVL sera previously tested for antibodies to L. infantum by DAT, showing a sensitivity of 93.1% and a specificity of 77.4%. The blotting test also showed a favorable band to detect visceral leishmaniasis. INTERPRETATION & CONCLUSION: According to the results, this new antigen had acceptable potential in detecting VL positive cases once western blotting was utilized, but the ELISA test did not proceed as expected for detecting true negative cases, probably due to some optimization issues.The present study is a promising start.

Role of intrauterine administration of transfected peripheral blood mononuclear cells by GM-CSF on embryo implantation and pregnancy rate in mice.

One of the effective treatments in women with recurrent implantation failure (RIF) is the use of immune cells to facilitate embryo implantation. Previous studies have shown that intrauterine transmission of peripheral blood mononuclear cells (PBMC) increased the embryo implantation rate. In this study using B6D2F1 (C57BL/6 × DBA2) mice, a fragment of the granulocyte macrophage colony-stimulating factor (Gm-csf) gene was cloned into an enhanced green fluorescent protein vector (pEGFP-N1) and then transfected into PBMC. The protein level of GM-CSF was evaluated in the transfected PBMC and untransfected PBMC by ELISA. Attachment of mouse embryos and the mRNA expression levels of leukemia inhibitory factor (Lif), vascular endothelial growth factor (Vegf), matrix metalloproteinase 9 (Mmp9), Gmcsf-receptor (Gmcsf-r) and interleukin 6 (Il6) in vitro were assessed by real-time PCR in endometrial cells. To determine the pregnancy rate and number of implantation sites in vivo, the mouse uterine horns were analyzed on Day 7.5 post coitum. A greater amount of GM-CSF was produced in PBMC transfected with recombinant vector (552 pg/mL) compared with the untransfected PBMC (57 pg/mL) and PBMC transfected with empty vector (34 pg/mL) (P < 0.05). The data showed that the embryo attachment rate and mRNA expression levels (Vegf [1.7-fold], Mmp9 [1.4-fold], Lif [1.5-fold], Gm-csf r [1.6-fold] and Il6 [1.2-fold]) in the in vitro study (P < 0.01), pregnancy rate (P < 0.01) and number of implantation sites (P < 0.01) in the in vivo investigation (P < 0.05) were increased in PBMC transfected with recombinant vector compared with the PBMC group. The study demonstrated that, in mice, endometrium immunotherapy with transfected PBMC that contained recombinant GM-CSF before embryo implantation was effective in improving embryo implantation and endometrial receptivity.

MicroRNA-218 competes with differentiation media in the induction of osteogenic differentiation of mesenchymal stem cell by regulating ß-catenin inhibitors.

Osteoporosis, a systemic skeletal disorder specified by low bone mass, is associated with bone fragility and the raised risk of fractures. Activation of the Wnt/ß-catenin signaling pathway has been directly demonstrated as a prominent biological event in the prevention of osteoporosis. Recently, critical roles of microRNAs (miRNAs) were further revealed in Wnt/ß-catenin signaling activation and thereby contributing to the development and maintenance of the human skeleton. In this study, we investigated whether miR-218 can significantly promote the osteogenic differentiation of mesenchymal stem cells in conditional media by regulating ß-catenin signaling inhibitors. The pre-miRNA nucleotide sequence of miR-218 was cloned into the pEGP-miR vector. Next, human adipose tissue-derived mesenchymal stem cells (AD-MSCs) were isolated, characterized, and transfected using pEGP-miR-218.Subsequently, the osteogenic potential of AD-MSCs was investigated in different treated groups using alkaline phosphatase (ALP)activity, calcium mineral deposition, and the expression of osteogenesis-related genes. Finally, negative regulators of Wnt signaling targeted by miR-218 were bioinformatically predicted. Our results indicated a significant increase in the ALP activity, mineralization, and osteogenesis-related genes expression in the AD-MSCs transfected with pEGP-miR-218. Also, the bioinformatic surveys and gene expression results showed that adenomatosis polyposis coli (APC) and glycogen synthase kinase 3 (GSK3-ß) were downregulated in the transfected AD-MSCs in both differential and conditional media. This study provided evidence that miR-218 can promote osteogenic differentiation of AD-MSCs even in conditional media. Therefore, our findings suggest miR-218 as a putative novel therapeutic candidate in the context of osteoporosis and other bone metabolism-related diseases.

Intraovarian injection of miR-224 as a marker of polycystic ovarian syndrome declines oocyte competency and embryo development.

miR-224 is associated with polycystic ovary syndrome (PCOS) that is an epidemic in reproductive age women. Most studies of miR-224 have focused on in vitro analyses, whereas the in vivo effects are not widely understood. In this study, we have conducted in silico analysis and found two potential miR-224 target genes, Ptx3 and Smad4 that have roles in folliculogenesis. Because patients with PCOS have decreased numbers of follicular cells related to cell apoptosis, we also investigated two apoptotic genes, Bax and Bcl2. We used the intraovarian injection method to deliver miR-224 into a mouse model. Histological examination of the ovaries was done by fluorescent microscope. Fertilization, cleavage, and developmental competence rates were counted under a stereomicroscope and compared between the studied groups. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of miR-224 was conducted to determine the levels of the studied genes in the oocytes, cumulus cells, and blastocysts. The numbers of oocytes and fertilization rate indicated a higher apoptosis index ( p < 0.05) and increased numbers of degenerated embryos with irregular blastomeres and fragmented cytoplasm in the experimental group. RT-PCR results indicated a significant increase in miR-224 levels in the manipulated group. Of the four analyzed genes, Ptx3, Smad4, and Bcl2 had decreased levels in the transfected group, with increased Bax expression ( p < 0.05). This data showed that miR-224 negatively affected ovulation in the mouse model by decreasing Ptx3 and Smad4 expressions. The changes in Bcl2 and Bax expression levels, as apoptosis biomarkers, showed that apoptosis was a secondary outcome of the effect of miR-224.

Generation of insulin-producing hepatocyte-like cells from human Wharton's jelly mesenchymal stem cells as an alternative source of islet cells.

Islet cell transplantation, as a treatment of type 1 diabetes, has a lot of complexity such as allograft rejections and an insufficient number of donors. The liver can be used as a replacement for endogenous insulin production. Hepatocytes can inherently respond to glucose levels and secrete proteins. Utilization of mesenchymal stem cells for curing diabetes represents a major focus of recent investigations. As a new choice for transplantation, we have proposed glucose-regulated insulin-producing hepatocyte-like cells, which produce insulin dependent on glucose levels. We have transfected human Wharton's jelly mesenchymal stem cells with the special construct, which included homology arms and glucose-responsive elements upstream of the minimum liver-type pyruvate kinase promoter-directed insulin gene. Then, we have differentiated these transfected cells to hepatocyte-like cells by using serial exposure of different inducing material and exogenous growth factors. Immunofluorescence analyses have demonstrated the expression of albumin, cytokeratin-18, Hep-Par1, α-fetoprotein, and insulin. The expression of hepatocyte marker genes in the differentiated cells was confirmed by reverse-transcription polymerase chain reaction. Interestingly, flow cytometry results showed that approximately 60% of the insulin-producing hepatocyte-like cells were simultaneously cytochrome P450 3A4 (CYP3A4) and insulin positive. CYP3A4 is a significant enzyme found in mature liver tissue. This confirmed that the differentiation and the transfection procedures were done correctly. They were functionally active by releasing insulin in response to elevated glucose concentrations in vitro. These applicable cells could be used in the liver for cell therapy of diabetes.

Transient knockdown of Nucleoside transporter 4 gene expression as a therapeutic target in Leishmania major by antisense RNA: In vitro and in vivo studies.

BACKGROUND & OBJECTIVES: Leishmania parasites cause various clinical symptoms in humans such as cutaneous ulcers and fatal visceral diseases. These parasites cannot synthesize purine rings de novo and must uptake purines from their hosts via salvage. Salvage is regulated by permeases in the cell membrane. There are hundreds of membrane transporter proteins to receive nutrients in Leishmania. Nucleoside transporter 4 (NT4) is one of the purine transporters that is involved in enhancing the uptake of adenine in Leishmania major. They are important new drug targets for the treatment of leishmaniasis because they can be used to transport toxic purine analogs to kill parasitic cells, thus preventing the progression of the infection. The present study was conducted to silence the NT4 nucleobase involved in the salvage pathway to interrupt purine nucleotide membrane transport in the cells of L. major. METHODS: In this study, a 502 bp segment of NT4 gene sequence was selected and designed as antisense transcripts after insertion in the parasite. The NT4 construct was transfected into L. major promastigotes for in vitro study of gene expression. Then, BALB/c mice infected with transgenic Leishmania and wild-type strain along with the number and size of lesions were studied in vivo. RESULTS: The study showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20. The percentages and the number of amastigotes in infected macrophages with wild-type strain L. major were more than infected macrophages with mutant parasites. Infected BALB/c mice with transgenic Leish- mania showed a lower number and size of lesions than the BALB/c mice infected with wild-type strain. INTERPRETATION & CONCLUSION: The results of the study indicated that the use of antisense RNA reduced NT4 gene expression in L. major. Further, studies are needed to ascertain that the use of antisense can be considered as a new treatment for leishmaniasis.

14-3-3ζ protein protects against brain ischemia/reperfusion injury and induces BDNF transcription after MCAO in rat.

Brain ischemia is a leading cause of death and disability worldwide that occurs when blood supply of the brain is disrupted. Brain-derived neurotrophic factor (BDNF) is a protective factor in neurodegenerative conditions. Nevertheless, there are some problems when exogenous BDNF is to be used in the clinic. 14-3-3ζ is a pro-survival highly-expressed protein in the brain that protects neurons against death. This study evaluates 14-3-3ζ effects on BDNF transcription at early time point after ischemia and its possible protective effects against ischemia damage. Human 14-3-3ζ protein was purified after expression. Rats were assigned into four groups, including sham, ischemia, and two treatment groups. Stereotaxic cannula implantation was carried out in the right cerebral ventricle. After one week, rats underwent middle cerebral artery occlusion (MCAO) surgery and received 14-3-3ζ (produced in our laboratory or standard form as control) in the middle of ischemia time. At 6 h of reperfusion after ischemia, brain parts containing the hippocampus, the cortex, the piriform cortex-amygdala and the striatum were collected for real time PCR analysis. At 24 h of reperfusion after ischemia, neurological function evaluation and infarction volume measurement were performed. The present study showed that 14-3-3ζ could up-regulate BDNF mRNA at early time point after ischemia in the hippocampus, in the cortex and in the piriform cortex-amygdala and could also improve neurological outcome and reduce infarct volume. It seems that 14-3-3ζ could be a candidate factor for increasing endogenous BDNF in the brain and a potential therapeutic factor against brain ischemia.