Correction to: Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

After publication of this paper, the authors determined an error in Fig. 4. Below is the correct Fig. 4.

Distinct effect of fetal bovine serum versus follicular fluid on multipotentiality of human granulosa cells in in vitro condition.

This study aimed to develop an appropriate medium for preservation of multipotentiality in human granulosa cells. To compare the possible effect of different media supplemented with follicular fluid or fetal bovine serum, granulosa cells were cultured in vitro over a period of 14 days. Stemness feature and any alteration in the cell phenotype were monitored using colony count assay and flow cytometry analysis by monitoring the expression of Oct3/4 and GATA-4 factors. Transcript expression level of Sox-2, Klf-4, and Nanog were investigated using quantitative real-time PCR analysis. Cells were cultured in the medium supplement with follicular fluid showed normal cell morphology and epithelial-like appearance, however, cells treated with fetal bovine serum, exhibited the clonogenic potential of granulosa cells which was increased after exposure to follicular fluid after 14 days (p < 0.05). Flow cytometry analysis revealed a significant reduction in the protein level of GATA-4 in cells cultured in presence of follicular fluid compared with cells received fetal bovine serum (p < 0.001). Quantitative real-time PCR analysis disclosed reduction of Sox-2, Klf-4 and Nanog levels in cells exposed to fetal bovine serum. Our experiment showed the exposure of human granulosa cells to follicular fluid efficiently preserves the stemness characteristics of the cells.

Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80 J/cm2, the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24 h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

Type 2 Diabetes Inhibited Human Mesenchymal Stem Cells Angiogenic Response by Over-Activity of the Autophagic Pathway.

The current study aimed to address the impact of serum from type 2 diabetes patients on the angiogenic properties of human bone marrow mesenchymal stem cells and its relationship to autophagy signaling. Human primary stem cells were enriched and incubated with serum from diabetic and normal subjects for 7 days. Compared to data from the control group, diabetic serum was found to induce a higher cellular death rate (P < 0.001) and apoptotic changes (P < 0.01). We also showed that diabetic condition significantly abolished angiogenesis tube formation on Matrigel substrate, decreased cell chemotaxis (P < 0.01) in response to SDF-1α, and inhibited endothelial differentiation rate (P < 0.0001). Western blotting showed autophagic status by high levels of P62 (P < 0.0001), beclin-1 (P < 0.0001), and increase in LC3II/I ratio (P < 0.001). In vivo Matrigel plug assay revealed that supernatant conditioned media prepared from cells exposed to diabetic serum caused a marked reduction in the recruitment of VE-cadherin- (P < 0.01) and α-SMA-positive (P < 0.0001) cells 7 days after subcutaneous injection. PCR expression array analysis confirmed the overexpression of autophagy and apoptosis genes in cultured cells in response to a diabetic condition (P < 0.05). Using bioinformatic analysis, we noted a crosstalk network between DM2, angiogenesis, and autophagy signaling. DM2 could potently modulate angiogenesis by the interaction of IL-1ß with downstream insulin receptor and upstream androgen receptor. Corroborating to data, diabetic serum led to abnormal regulation of P62 during the angiogenic response. These data demonstrate that diabetic serum decreased human mesenchymal stem cell angiogenic properties directly on angiogenesis pathways or by the induction of autophagy signaling. J. Cell. Biochem. 118: 1518-1530, 2017. © 2016 Wiley Periodicals, Inc.

Magnetic layered double hydroxides for the sensitive dispersive solid phase microextraction of hippuric acid in urine samples prior to HPLC-UV analysis.

The core- shell structural layered double hydroxide (Fe3O4-SiO2-EN@Zn-Al-LDH) was successfully synthesized and applied as a solid sorbent in the magnetic dispersive micro solid-phase-extraction (M-DµSPE) method. It was combined with high-performance liquid chromatography for the trace analysis of hippuric acid (HA) from urine samples. The obtained magnetic layered double hydroxides (LDHs) were characterized by XRD, FT-IR, VSM, FE-SEM, and BET techniques. The characterization analysis indicated that Fe3O4- SiO2- EN@ Zn-Al-LDH has a sufficient surface area and good saturation magnetism. The affecting variables on the extraction of HA by the proposed method were optimized. Excellent adsorption capacity (127.8 mg g-1), wide linearity dynamic range (0.015-500 µg mL-1), and satisfactory limits of detection and quantification (0.055 and 0.014 µg mL-1, respectively) could be obtained under optimum conditions. The good repeatability and low relative standard deviation (7.2 %), low carry-over (2.7%), good matrix effect (93.6%), high reusability (up to 19 times), and an acceptable percent recovery value (97.2%) proved the selectivity and applicability of the proposed method for the extraction of the trace levels of HA in real urine samples.

Modulatory Effect of Vitamin C on Hypoxia Induced Breast Cancer Stem Cells.

Purpose: Eliminating cancer stem cells (CSCs) is a challenge because of their enhanced resistance to anti-cancer drugs. Vitamin C, which is insufficient in patients with higher stages of cancer, has been gaining attention as a potential treatment for human malignancies. Hence this study aimed to analyze the effect of high-dose vitamin C treatment on the gene expression level of HIF-1α, NF-κB1, BAX, and DNMT1 in the MCF7 cells undergoing hypoxia, as an inducer of CSCs characteristics. As a result, vitamin C could be possibly used as a promising therapeutic adjuvant. Methods: Here we first analyzed the breast CSC population alteration in MCF7 cells following hypoxia induction. Then, we evaluated the impact of vitamin C treatment on the gene expression level of four stemness-related genes in hypoxic MCF7 cells. Results: Our results indicate that vitamin C could reduce proliferation and stemness states in CSCs possibly by induction of apoptotic markers such as BAX, along with attenuating stemness markers, including NF-κB1, and DNMT1 gene expressions. Conclusion: According to our findings, vitamin C administration would become a new approach to avoiding the stimulation of CSCs during cancer therapies.

Downregulation of TdT Expression through Splicing Modulation by Antisense Peptide Nucleic Acid (PNA).

BACKGROUND AND OBJECTIVE: Antisense oligonucleotides are able to modulate splicing patterns and offer therapeutic intervention for cancer and other diseases. Considering TdT potential as a target in cancer therapy, the present study aimed to investigate splicing alteration of TdT pre-mRNA in Molt-4 cells using peptide nucleic acid (PNA) octaarginine and cholic acid conjugates. METHOD: We examined 16 mer PNAs targeting 5' and 3' junctions of intron 7 and addressed their mRNA splicing modulation effects using RT-PCR analysis. We also tested corresponding 2-base mismatch PNAs to confirm the sequence specificity. In addition, protien level of TdT, apoptosis induction and cell viability rate were analysed. RESULTS: PCR analysis showed that full match PNAs could modulate the splicing process, thereby producing a longer mRNA still including intron 7. PCR results also implied exon 7 skipping. In addition, reduced level of TdT protein in Molt-4 cells was observed. Downregulation of TdT level in PNA treated cells was accompanied by an increased rate of apoptosis and decreased the level of cell survival. CONCLUSION: PNA-mediated splicing modulation can specifically downregulate TdT expression. TdT dowregulation results in apoptosis induction and reduced cell survival in Molt-4 cells. These observations could draw more attentions to develop PNA based strategies for TdT suppression and consequent apoptosis induction in acute lymphoblastic leukemia.

Impact of morphine on the expression of insulin receptor and protein levels of insulin/IGFs in rat neural stem cells.

Alzheimer's disease is correlated with neuronal degeneration and loss of neuronal precursors in different parts of the brain. It has been found disturbance in the homeostasis neural stem cells (NSCs) can cause neurodegeneration. Morphine, an analgesic agent, can disrupt the dynamic and normal state of NSCs. However, more investigations are required to clearly address underlying mechanisms. The current experiment aimed to investigate the effects of morphine on the cell distribution of insulin factor and receptor and insulin-like growth factors (IGF1, IGF2) in NSCs. NSCs were isolated from rats and stemness feature confirmed by antibodies against nestin and Sox2. The cells were exposed to 100µM morphine, 50µM naloxone and combination of these two drugs for 72h. The neural cell growth, changes in levels of insulin and insulin-like growth factors secreted by NSCs as well as the insulin-receptor-gene expression were assessed by flow cytometry, ELlSA, and real-time PCR, respectively. Cell cycle assay revealed the exposure of cells to morphine for 72h increased cell apoptosis and decreased neural stem cell growth. The biosynthesis of insulin, insulin-like growth factors, and insulin receptor were reduced (p<0.05) after NSCs exposure to morphine at the concentration of 100µM for 24, 48 and 72h. Naloxone is a competitive antagonist which binds MOR where morphine (and endogenous opioids) bind, and reversed the detrimental effects of morphine. It can be concluded that morphine initiated irregularity in NSCs kinetics and activity by reducing the secretion of insulin and insulin-like growth factors and down-regulation of insulin receptor.

Bone marrow mesenchymal stem cells and their conditioned media could potentially ameliorate ovalbumin-induced asthmatic changes.

BACKGROUND: The major feature of asthma is governed by chronic airway inflammation. This investigation was proposed to achieve the suitable candidate for ameliorating long-term chronic asthmatic changes of respiratory tract. METHODS: 36 rats were classified into healthy (C) and ovalbumin (OVA)-sensitized animals (S). To sensitize, the rats were exposed to OVA over a course of 32±1days. One day after sensitization, equal six different groups were subjected to experimental procedure (n=6); Rats only received intratracheally 50ml PBS (CPT and SPT groups), 50µl conditioned medium (CM) (CST and SST groups) and 50µl PBS containing 2×106 rat bone marrow-derived mesenchymal stem cells (rBMMSCs) (CCT and SCT groups). Two weeks after treatment, tracheal responsiveness, immunologic responses and recruitment of rBMMSCs into the lung as well as pathological changes were evaluated. RESULTS: A high degree of tracheal responsiveness, total white blood cell and percentages of eosinophil and neutrophil was significantly recorded in all sensitized groups rather than of controls (p<0.001 to p<0.05). Of interest, all above-mentioned parameters decreased significantly in SST and notably SCT groups as compared to S group (p<0.001 to p<0.05). The results revealed decrease number of blood CD3+CD4+ and concurrent increase in CD3+CD8+ in all sensitized rats as compared to control (p<0.001 to p<0.05). Noticeably, no significant modulatory effects of either cell or CM administration were achieved on the CD3+CD4+ and CD3+CD8+ populations in non-asthmatic rats. Moreover, the number of CD3+CD4+ in SST and SCT groups tended to increase, which coincided with a decreased manner of CD3+CD8+ populations as compared with S group (p<0.001 to p<0.05). However, the CD3+CD4+ cells in SCT rats were significantly higher than the group SST (p<0.01) whereas CD3+CD8+ cells diminished simultaneously (p<0.001). Real-time PCR analysis further showed that both CM and particularly MSCs changed the expression of interleukin (IL)-4 and IL-10 in the asthmatic groups to the near level of control rats (p<0.001 to p<0.05). Histopathological analysis revealed a profound reduction of lungs injuries in asthmatic rats when received CM and peculiarly mesenchymal stem cells (p<0.01 to p<0.05). CONCLUSION: Our study shed light on the superior effects of rBMMSCs, rather than CM, in attenuating of chronic asthmatic changes in the rat model.

The role of morphine on rat neural stem cells viability, neuro-angiogenesis and neuro-steroidgenesis properties.

A lack of comprehensive data exists on the effect of morphine on neural stem cell neuro-steroidogenesis and neuro-angiogenesis properties. We, herein, investigated the effects of morphine (100µM), naloxone (100µM) and their combination on rat neural stem cells viability, clonogenicity and Ki-67 expression over a period of 72h. Any alterations in the total fatty acids profile under treatment protocols were elucidated by direct transesterification method. We also monitored the expression of p53, aromatase and 5-alpha reductase by real-time PCR assay. To examine angiogenic capacity, in vitro tubulogenesis and the level of VE-cadherin transcript were investigated during neural to endothelial differentiation under the experimental procedure. Cells supplemented with morphine displayed reduced survival (p<0.01) and clonogenicity (p<0.001). Flow cytometric analysis showed a decrease in Ki-67 during 72h. Naloxone potentially blunted morphine-induced all effects. The normal levels of fatty acids, including saturated and unsaturated were altered by naloxone and morphine supplements. Following 48h, the up-regulation of p53, aromatase and 5-alpha reductase genes occurred in morphine-primed cells. Using three-dimensional culture models of angiogenesis and real time PCR assay, we showed morphine impaired the tubulogenesis properties of neural stem cells (p<0.001) by the inhibition of trans-differentiation into vascular cells and led to decrease of in VE-cadherin expression. Collectively, morphine strongly impaired the healthy status of neural stem cells by inducing p53 and concurrent elevation of aromatase and 5-alpha reductase activities especially during early 48h. Also, neural stem cells-being exposed to morphine lost their potency to elicit angiogenesis.

Copper sulfate pentahydrate reduced epithelial cytotoxicity induced by lipopolysaccharide from enterogenic bacteria.

The over usage of multiple antibiotics contributes to the emergence of a whole range of antibiotic-resistant strains of bacteria causing enterogenic infections in poultry science. Therefore, finding an appropriate alternative natural substance carrying an antibacterial capacity would be immensely beneficial. It has been previously discovered that the different types of cupric salts, especially copper sulfate pentahydrate (CuSO4·5H2O), to carry a potent bactericidal capacity. We investigated the neutralizing effect of CuSO4·5H2O (6.25µg/ml) on the reactive oxygen species generation, and expression of MyD88, an essential adaptor protein of Toll-like receptor, and NF-κB in three intestinal epithelial cell lines exposed to 50ng/ml lipopolysaccharide. In order to find the optimal cupric sulfate concentration without enteritis-inducing toxicity, broiler chickens were initially fed with water containing 0.4, 0.5, and 1mg/l during a period of 4days. After determination of appropriate dosage, two broiler chickens and turkey flocks with enteritis were fed with cupric compound for 4days. We found that cupric sulfate can lessen the cytotoxic effect of lipopolysaccharide by reducing the reactive oxygen species content (p<0.05). Additionally, the expression of MyD88 and NF-κB was remarkably down-regulated in the presence of lipopolysaccharide and cupric sulfate. The copper sulfate in doses lower than 0.4mg/ml expressed no cytotoxic effect on the liver, kidney, and the intestinal tract while a concentration of 0.5 and 1mg/ml contributed to a moderate to severe tissue injuries. Pearson Chi-Square analysis revealed the copper cation significantly diminished the rate of mortality during 4-day feeding of broiler chicken and turkey with enteritis (p=0.000). Thus, the results briefed above all confirm the potent anti-bactericidal feature of cupric sulfate during the course of enteritis.

Evaluation of the Effect of Ascorbic Acid Administration on Gene Expression Level of IL-6 and TNF-α Cytokines in Deceased Donors.

Brain death is associated with increased inflammatory cytokines levels and poor graft quality to transplant. We aimed to evaluate the impact of Ascorbic Acid (AA) on the inflammatory status of Brain-Dead Donors (BDDs). Forty BDDs were randomly divided into two groups. Donor treatment (n=20) consisted of 100 mg/kg AA infusion 6 hours before donor operation and subsequent infusion of 100 mg/kg/p6h until organ removal. Blood samples were taken at three times, 6 hours before donor surgery (TP(1)), immediately after laparotomy (TP(2)), and before organ removal (TP(3)). Gene expression level and serum concentration of IL-6 and TNF-α cytokines were assessed by real-time PCR and ELISA methods. To investigate transplanted liver function, serum values of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), and Billirubin-Total were evaluated on the 1(st), 3(rd), and 10(th) postoperative days. We found a significant reduction in IL-6 mRNA expression ratio of TP(3) to TP(1) following AA application among BDDs. Despite the considerable decrease in treated donors regarding IL-6 mRNA expression ratio of TP(2) to TP(1), TP(3) to TP(2), and also TNF-α variations in these periods, the results were not significant. Regarding serum concentration of these cytokines, particularly IL-6, there was a decrease between TP(2) and TP(3) following AA application in the treated donors. Furthermore, a significant reduction was found in serum AST and ALT levels in the recipients of treated group on the 3(rd) day compared to the 1(st) day after transplantation. It seems that AA beneficially affects the inflammatory status of BDDs, resulting in improved primary allograft function.