Electrospun triple-layered PLLA/gelatin. PRGF/PLLA scaffold induces fibroblast migration.

The function of fibroblast cells in wounded areas results in reconstruction of the extra cellular matrix and consequently resolution of granulation tissue. It is suggested that the use of platelet-rich plasma can accelerate the healing process in nonhealing or slow-healing wounds. In this study, a simple and novel method has been used to fabricate an electrospun three-layered scaffold containing plasma rich in growth factor with the aim of increasing the proliferation and migration of fibroblast cells in vitro. First, plasma rich in growth factor was derived from platelet rich plasma, and then a three-layered scaffold was fabricated using PLLA nanofibers as the outer layers and plasma rich in growth factor-containing gelatin fibers as the internal layer. The growth morphology of cells seeded on this scaffold was compared to those seeded on one layered PLLA scaffold. The study of the cell growth rate on different substrates and the migration of cells in response to the drug release of multilayered scaffold was investigated by the cell quantification assay and a modified under agarose assay. Scanning electron microscopy and fluorescence images showed that cells seeded on multilayered scaffold were completely oriented 72 hours after seeding compared to those seeded on PLLA scaffold. The cell quantification assay also indicated significant increase in proliferation rate of cells seeded on three-layered scaffold compared to those seeded on PLLA scaffold and finally, monitoring cell migration proved that cells migrate significantly toward the three-layered scaffold up to 48 to 72 hours and afterwards start to show a diminished migration rate toward this scaffold.

The effect of bone marrow mesenchymal stromal cell exosomes on acute myeloid leukemia's biological functions: a focus on the potential role of LncRNAs.

Acute myeloid leukemia represents a group of malignant blood disorders that originate from clonal over-proliferation and the differentiation failure of hematopoietic precursors, resulting in the accumulation of blasts in the bone marrow. Mesenchymal stromal cells (MSCs) have been shown to exert diverse effects on tumor cells through direct and indirect interaction. Exosomes, as one of the means of indirect intercellular communication, are released from different types of cells, including MSCs, and their various contents, such as lncRNAs, enable them to exert significant impacts on target cells. Our study aims to investigate the effects of BM-MSC exosomes on the cellular and molecular characterization of HL-60 AML cells, particularly detecting the alterations in the expression of lncRNAs involved in AML leukemogenesis, cell growth, drug resistance, and poor prognosis. BM-MSCs were cultured with serum-free culture media to isolate exosomes from their supernatants. The validation of exosomes was performed in three stages: morphological analysis using TEM, size evaluation using DLS, and CD marker identification using flow cytometry. Subsequently, the HL-60 AML cells were treated with isolated BM-MSC exosomes to determine the impact of their contents on leukemic cells. Cell metabolic activity was evaluated by the MTT assay, while cell cycle progression, apoptosis, ROS levels, and proliferation were assessed by flow cytometry. Furthermore, RT-qPCR was conducted to determine the expression levels of lncRNAs and apoptosis-, ROS-, and cell cycle-related genes. MTT assay and flow cytometry analysis revealed that BM-MSC exosomes considerably suppressed cell metabolic activity, proliferation, and cell cycle progression. Also, these exosomes could effectively increase apoptosis and ROS levels in HL-60 cells. The expression levels of p53, p21, BAX, and FOXO4 were increased, while the BCL2 and c-Myc levels decreased. MALAT1, HOTAIR, and H19 expression levels were also significantly decreased in treated HL-60 cells compared to their untreated counterparts. BM-MSC exosomes suppress cell cycle progression, proliferation, and metabolic activity while simultaneously elevating the ROS index and apoptosis ratio in HL-60 cells, likely by reducing the expression levels of MALAT1, HOTAIR, and H19. These findings suggest that BM-MSC exosomes might serve as potential supportive therapies for leukemia.

Purification, and characterization of a new pro-coagulant protein from Iranian Echis carinatus venom.

This work aimed to purify the proteins that cause blood coagulation in the venom of the Iranian Echis carinatus snake species in a comprehensive manner. Gel filtration chromatography (GFC), Ion exchange chromatography (IEC), and Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC) were utilized in the purification of the coagulation factors. The prothrombin clotting time (PRCT) and SDS-PAGE electrophoresis were performed to confirm the coagulative fractions. The fraction with the shortest coagulation time was selected. The components of this designated fraction were identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) following thorough purification. Circular dichroism (CD) was employed to determine the second structure of the coagulation factor. The crude venom (CV) was analyzed and had a total protein concentration of 97%. Furthermore, the PRCT of the crude venom solution at a concentration of 1 mg/ml was determined to be 24.19 ± 1.05 s. The dosage administered was found to be a factor in the venom's capacity to induce hemolysis. According to CD analysis, the protein under investigation had a helical structure of 16.7%, a beta structure of 41%, and a turn structure of 9.8%. CHNS proved that the purified coagulant protein had a Carbon content of 77.82%, 5.66% Hydrogen, 3.19% Nitrogen, and 0.49% Sulphur. In the present investigation, a particular type of snake venom metalloproteinase (SVMP) has undergone the process of purification and characterization and has been designated as EC-124. This purified fraction shows significant efficacy as a procoagulant. Our findings have shown that this compound has a function similar to factor X and most likely it can cause blood coagulation by activating factor II (FII).

Expression Changes of SIRT1 and FOXO3a Significantly Correlate with Oxidative Stress Resistance Genes in AML Patients.

The increased metabolism in acute myeloid leukemia (AML) malignant cells resulted in the production of high levels of free radicals, called oxidative stress conditions. To avoid this situation, malignant cells produce a considerable amount of antioxidant agents, which will lead to the release of a continuous low level of reactive oxygen species (ROS), causing genomic damage and subsequent clonal evolution. SIRT1 has a key role in driving the adaptation to this condition, mainly through the deacetylation of FOXO3a that affects the expression of oxidative stress resistance target genes such as Catalase and Manganese superoxide dismutase (MnSOD). The aim of this study is to simultaneously investigate the expression of SIRT1, FOXO3a, and free radical-neutralizing enzymes such as Catalase and MnSOD in AML patients and measure their simultaneous change in relation to each other. The gene expression was analyzed using Real Time-PCR in 65 AML patients and 10 healthy controls. Our finding revealed that expression of SIRT1, FOXO3a, MnSOD and Catalase was significantly higher in AML patients in comparison to healthy controls. Also, there was a significant correlation between the expression of SIRT1 and FOXO3a, as well as among the expression of FOXO3a, MnSOD and Catalase genes in patients. According to the results, the expression of genes involved in oxidative stress resistance was higher in AML patients, which possibly contributed to the development of malignant clones. Also, the correlation between the expression of SIRT1 and FOXO3a gene reflects the importance of these two genes in increased oxidative stress resistance of cancer cells.

Platelet MicroRNA-484 as a Novel Diagnostic Biomarker for Acute Coronary Syndrome.

OBJECTIVE: Platelet microRNAs (miRs) have been indicated as a diagnostic biomarker in various diseases, including acute coronary syndrome (ACS). This study aimed to investigate the expression of miR-223-5p, miR-126-5p, miR-484, and miR-130a-3p in individuals with coronary artery disease (CAD). METHODS: Forty subjects with CAD and 13 healthy individuals were under study. The expression of miR-223-5p, miR-126-5p, miR-484, and miR-130a-3p was measured in platelets by quantitative reverse transcription-polymerase chain reaction. The relationship between miRNA expression and various parameters of the subjects was analyzed using analysis of variance and Spearman and t-tests. RESULTS: The miR-484 expression was significantly upregulated in the ACS subjects (P = .0097). Moreover, miR-484 had diagnostic value for screening subjects with unstable angina vs controls (area under the curve [AUC] = 0.978, 95% confidence interval [CI] 0.92-1, P = .0006) and NSTEMI patients versus controls (AUC = 0.910, 95% CI 0.74-1, P = .005). CONCLUSION: The results of this study indicate that the upregulated expression of miR-484 in ACS patients might be used as a diagnostic biomarker in ACS.

Morphological, cytotoxicity, and coagulation assessments of perlite as a new hemostatic biomaterial.

Hemorrhage control is vital for clinical outcomes after surgical treatment and pre-hospital trauma injuries. Numerous biomaterials have been investigated to control surgical and traumatic bleeding. In this study, for the first time, perlite was introduced as an aluminosilicate biomaterial and compared with other ceramics such as kaolin and bentonite in terms of morphology, cytotoxicity, mutagenicity, and hemostatic evaluations. Cellular studies showed that perlite has excellent viability, good cell adhesion, and high anti-mutagenicity. Coagulation results demonstrated that the shortest clotting time (140 seconds with a concentration of 50 mg mL-1) was obtained for perlite samples compared to other samples. Therefore, perlite seems most efficient as a biocompatible ceramic for hemorrhage control and other biomaterial designs.

Plasma Cell Proliferation Is Reduced in Myeloma-Induced Hypercalcemia and in Co-Culture with Normal Healthy BM-MSCs.

OBJECTIVE: In multiple myeloma (MM), stimulation of osteoclasts and bone marrow (BM) lesions lead to hypercalcemia, renal failure, and anemia. Co-culture of the myeloma cells in both hypocalcemia and hypercalcemia concentrations with bone marrow-mesenchymal stem cells were evaluated. MATERIALS AND METHODS: Viability and survival of myeloma cells were assessed by microculture tetrazolium test and flow cytometric assays. Mesenchymal stem cells (MSCs) were extracted from normal and myeloma patients and were co-cultured with myeloma cells. RESULTS: Myeloma cells showed less survival in both hypocalcaemia and hypercalcemia conditions (P <.01). The paracrine and juxtacrine conditions of demineralized bone matrix-induced hypercalcemia increased the proliferation and survival of the cells (P <.05). Unlike myeloma MSCs, normal MSCs reduced the survival of and induced apoptosis in myeloma cells (P <.1). CONCLUSION: Normal healthy-MSCs do not protect myeloma cells, but inhibit them. However, increasing the ratio of myeloma cells to MSCs reduces their inhibitory effects of MSCs and leads to their myelomatous transformation.

Investigating the inhibitory effect of miR-34a, miR-449a, miR-1827, and miR-106b on target genes including NOTCH1, c-Myc, and CCND1 in human T cell acute lymphoblastic leukemia clinical samples and cell line.

OBJECTIVES: microRNAs are small non-coding molecules that regulate gene expression in various biological processes. T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy accompanied with genetic aberrations and accounts for 20% of children's and adult's ALL. Notch signaling pathway dysregulation occurs in 60% of T-ALL cases. In the present study, we aimed to determine the relationship between miRNAs and genes involved in Notch signaling pathway. MATERIALS AND METHODS: Considering the role of the pathway and its down-stream genes in proliferation, differentiation, cell cycle, and apoptosis, NOTCH1, c-Myc, and CCND1 genes were selected as target genes. Using bioinformatics studies, miR-34a, miR-449a, miR-1827, and miR-106b were selected as miRNAs targeting the above-mentioned genes. We evaluated these genes and miRNAs in T-ALL clinical samples as well as Jurkat cell line, in which NOTCH1 is overexpressed. RESULTS: Quantitative Real-Time PCR indicated that NOTCH1, c-Myc, and CCND1 were overexpressed in samples with decreased expression of miR-34a. In addition, we observed that samples with decreased expression of miR-449a showed increased expression of NOTCH1 and CCND1. Furthermore, we analyzed the expression of miR-1827 and miR-106b, which target c-Myc and CCND1, respectively. We found out that the expression of miR-1827, miR-106b, and their respective target genes were inversely correlated in 80% and 75% of the cases (r=0.8), respectively. Furthermore, in Jurkat cell line, the expression of target genes was increased while the candidate miRNAs except miR-34a were decreased. CONCLUSION: These miRNAs can be proposed as biomarkers and new therapeutic targets in T-ALL patients who have NOTCH1 overexpression.

Severe plasma prekallikrein deficiency: Clinical characteristics, novel KLKB1 mutations, and estimated prevalence.

BACKGROUND: Severe plasma prekallikrein (PK) deficiency is an autosomal-recessive defect characterized by isolated activated partial thromboplastin time prolongation. To date, no comprehensive methodologically firm analysis has investigated the diagnostic, clinical, and genetic characteristics of PK deficiency, and its prevalence remains unknown. PATIENTS/METHODS: We described new families with PK deficiency, retrieved clinical and laboratory information of cases systematically searched in the (gray) literature, and collected blood of these cases for complementary analyses. The Genome Aggregation Database (gnomAD) and the population-based Gutenberg Health Study served to study the prevalence of mutations and relevant genetic variants. RESULTS: We assembled a cohort of 111 cases from 89 families and performed new genetic analyses in eight families (three unpublished). We identified new KLKB1 mutations, excluded the pathogenicity of some of the previously described ones, and estimated a prevalence of severe PK deficiency of 1/155 668 overall and 1/4725 among Africans. One individual reported with PK deficiency had, in fact, congenital kininogen deficiency associated with decreased PK activity. One quarter of individuals had factor XII clotting activity below the reference range. Four major bleeding events were described in 96 individuals, of which 3 were provoked, for a prevalence of 4% and an annualized rate of 0.1%. The prevalence of cardiovascular events was 15% (6% <40 years; 21% 40-65 years; 33% >65 years) for an annualized rate of 0.4%. CONCLUSIONS: We characterized the genetic background of severe PK deficiency, critically appraised mutations, and provided prevalence estimates. Our data on laboratory characteristics and clinical course of severe PK deficiency may have clinical implications.

Expression Pattern of Neuronal Markers in PB-MSCs Treated by Growth Factors Noggin, bFGF and EGF.

Mesenchymal stem cells (MSCs) have the ability to differentiate into neuronal like cells under appropriate culture condition. In this study, we investigated whether MSCs derived from human peripheral blood (PB-MSCs) can differentiate into neuronal like cells by synergic effect of the growth factors EGF, bFGF and Noggin. For this purpose, the expression of five neuronal markers (Nestin, ß III tubulin, NFM, MAP2 and NSE) were evaluated in treated PB-MSCs by SYBR Green Real time PCR. The expression analysis showed a higher expression of ß-tubulin and NFM in treated BP-MSCs compared with untreated PB-MSCs as a control group. The expression of Nestin was also diminished in PB-MSCs treated with Noggin. This study suggested that the treatment of PB- MSCs with Noggin alongside with bFGF and EGF might differentiate these cells into neuronal lineage cells. The obtained results could be further developed for useful applications in regenerative medicine.