Bone regeneration in rat using polycaprolactone/gelatin/epinephrine scaffold.

Solid supports like the extracellular matrix network are necessary for bone cell attachment and start healing in the damaged bone. Scaffolds which are made of different materials are widely used as a supportive structure in bone tissue engineering. In the current study, a 3D polycaprolactone/gelatin bone scaffold was developed by blending electrospinning and freeze-drying techniques for bone tissue engineering. To improve the efficiency of the scaffold, different concentrations of epinephrine (EP) due to its effect on bone healing were loaded. Fabricated scaffolds were characterized by different tests such as surface morphology, FTIR, porosity, compressive strength, water contact angle, and degradation rate. The interaction between prepared scaffolds and blood and cells was evaluated by hemolysis, and MTT test, respectively, and bone healing was evaluated by a rat calvaria defect model. Based on the results, the porosity of scaffolds was about 75% and by adding EP, mechanical strength decreased while due to the hydrophilic properties of it, degradation rate increased. In vivo and in vitro studies showed the best cell proliferation and bone healing were in PCL/gelatin/EP1% treated group. These results showed the positive effect of fabricated scaffold on osteogenesis and bone healing and the possibility of using it in clinical trials.

Runx2 silencing promotes adipogenesis via down-regulation of DLK1 in chondrogenic differentiating MSCs.

BACKGROUND: For cartilage regeneration, stem cells are a promising cell source; however, even the advances made in the differentiation of stem cells into precursor-differentiated cartilage cells have not been successful with respect to reprograming these cells to achieve complete differentiation and fully functioning cells until now. Previous findings suggest that Runx2 plays a major role in chondrocyte differentiation and maturation. Although targeting Runx2 has enhanced some chondrocyte properties, the adipogenic lineage shift has eventually occurred in these cells. The present study mainly aimed to reveal the mechanism of this adipogenesis. METHODS: To create inducible artificial shRNA-miR expressing vectors, the designed short hairpin RNAs (shRNAs) were inserted into the pri-mir-30 backbone, cloned into lentiviral pLVET-Tet-on, and transducted into mesenchymal stem cells (MSCs). Runx2 gene was silenced in MSCs either for 1 week or 4 weeks and cultured in the chondrogenic medium. At days 7, 14 and 28, cells were harvested, and chondrogenesis, adipogenesis and hypertrophic states were examined using histochemical staining and a real-time polymerase chain reaction assay. RESULTS: The results showed that the designed shRNA-miR effectively targeted Runx2 in mRNA and protein levels. Chondrogenic markers were up-regulated in constantly silenced Runx2 group; however, adipogenic markers and fat droplets appeared gradually. DLK1 gene was also significantly down-regulated in this group, and overexpression of DLK1 abrogated adipogenesis in the Runx2 targeted group. CONCLUSIONS: Based on these results, it can be concluded that DLK1 is responsible for the lineage shift in Runx2 targeted chondrogenic differentiating MSCs.

Design of an engineered ACE2 as a novel therapeutics against COVID-19.

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.

Cadmium nanocluster as a safe nanocarrier: biodistribution in BALB/c mice and application to carry crocin to breast cancer cell lines.

Aim: Metal nanoclusters are emerging nanomaterials applicable for drug delivery. Here, the toxicity and oxidative stress induction of divalent cationic cadmium (Cd2+) was compared with a Cd in the form of nanocluster. Then, it was used for targeted drug delivery into breast cancer cell lines. Methods: Using a green chemistry route, a Cd nanocluster (Cd-NC) was synthesized based on bovine serum albumin. After characterization, its genotoxicity and oxidative stress induction were studied in both in vitro and in vivo. After that, it was conjugated with hyaluronic acid (HA). The efficiency of hyaloronized-Cd-CN (HA-Cd-NC) for loading and releasing crocin (Cro), an anticancer phytochemical, was studied. Finally, it was applied for cell death induction in a panel of breast cancer cell lines. Results: The comet assay results indicated that, unlike Cd2+ and potassium permanganate (KMnO4), no genotoxicity and oxidative stress was induced by Cd-NC in vitro. Then, the pharmacokinetics of this Cd-NC was studied in vivo. The data showed that Cd-NC has accumulated in the liver and excreted from the feces of mice. Unlike Cd2+, no toxicity and oxidative stress were induced by this Cd-NC in animal tissues. Then, the Cd-NC was targeted toward breast cancer cells by adding HA, a ligand for the CD44 cell surface receptor. After that, Cro was loaded on HA-Cd-NC and it was used for the treatment of a panel of human breast cancer cell lines with varying degrees of CD44. The half-maximal drug inhibitory concentration (IC50) of Cro was significantly decreased when it was loaded on HA-Cd-NC, especially in MDA-MB-468 with a higher degree of CD44 at the surface. These results indicate the higher toxicity of Cro toward breast cancers when carried out by HA-Cd-NC. Conclusions: The Cd-NC was completely safe and is a promising candidate for delivering anticancer drugs/phytochemicals into the targeted breast tumors.

Oleuropein as An Effective Suppressor of Inflammation and MicroRNA-146a Expression in Patients with Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a common progressive autoimmune disorder that causes chronic inflammation of the joints and damage to other organs. Previous studies have reported the important role of miRNA-146a in the pathogenesis of RA. In addition, the anti-inflammatory and modulatory effects of oleuropein (OLEU) on the expression pattern of microRNAs (miRNAs) have been shown in different diseases. Therefore, this study aimed to evaluate both the sensitivity and specificity of miRNA-146a and determine the potential effects of OLEU on the expression levels of miRNA-146a and tumour necrosis factor-alpha (TNF-α) in RA patients. MATERIALS AND METHODS: The participants in this experimental study were divided into 2 groups: RA (n=45) and healthy controls (n=30). The isolated peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of OLEU; and the level of TNF-α expression, anti-citrullinated protein, and miRNA-146a were determined using enzyme-linked immunoassay and real-time polymerase chain reaction, respectively. In addition, the receiver operating characteristic (ROC) curve analysis evaluated the sensitivity and specificity of miRNA-146a in RA patients. RESULTS: Results revealed a positive correlation between the levels of miRNA-146a expression with the serum levels of C-reactive protein (CRP) and rheumatoid factor (RF) in RA patients. In addition, OLEU treatment decreased the levels of TNF-α and miRNA-146a expression in treated PBMCs samples compared with untreated cells. The ROC curve analysis showed an 85% sensitivity and 100% specificity of miRNA-146a in RA patients. CONCLUSION: Therefore, miRNA-146a can be used as a useful biomarker for RA diagnosis, particularly for early detection. In addition, OLEU could suppress inflammation in RA patients through the regulation of miRNA-146a.

Aqueous-alcoholic Ferulla extract reduces memory impairments in rats exposed to cadmium chloride.

INTRODUCTION: Cadmium (Cd) is the most dangerous heavy metal that is becoming more widespread in nature as a result of industrial activities. One of the toxic effects of Cd on the body is its neurological effect. The mechanism of these effects has been attributed to the induction of oxidative stress. Ferulla plant has antioxidant properties. In the present study, the aim was to reduce the toxic effects of Cd on memory impairment in rats by through the consumption of Ferulla extract. MATERIALS & METHOD: Rats were randomly divided into five groups of six: (1) control group, (2) 300 µM cadmium exposure group, and three treatment groups with doses of (3) 100, (4) 300, and (5) 600 mg/kg.BW of F. Ferulla extract after Cd exposure. To induce neurotoxicity, Cd was daily injected peritoneally at a concentration of 300 µM in 1 ml of normal saline for a week. Next, for 3 weeks, the Cd group received 1 ml of normal peritoneal saline, and the treatment groups received F. Ferulla extract at concentrations of 100, 300, and 600 mg/kg.BW in 1 ml of normal saline daily for a week. At the end of the treatment period, a water maze was used to assess memory disorders. Malondialdehyde (MDA), glutathione concentration (GSH), and glutathione peroxidase (GPX) activity in nerve tissue were also measured. Morris water maze was also performed after intervention. RESULTS: Cd-induced neurotoxicity was shown in Cd groups. MDA, GSH, and GPX have a significant difference in comparison between the Cd and 300, 600 treated groups. MDA has a significant increase (p < 0.05), and GSH and GPX have a significant decrease (p < 0.05). The results of the Morris water maze showed that the Cd group spent either 300 or 600 more distances and time to find a place to escape, which was significant (p < 0.05) CONCLUSION: Cd exposure can induce neurotoxicity and disrupt learning and memory. On the other hand, Ferulla extract can improve learning and memory in Cd-induced neurotoxicity model via induced antioxidant defense system.

Fabrication and Characterization of Nanofibrous Poly (L-Lactic Acid)/Chitosan-Based Scaffold by Liquid-Liquid Phase Separation Technique for Nerve Tissue Engineering.

Fabrication method is one of the essential factors which directly affect on the properties of scaffold. Several techniques have been well established to fabricate nanofibrous scaffolds such as electrospinning. However, preparing a three-dimensional (3-D) interconnected macro-pore scaffold essential for transporting the cell metabolites and nutrients is difficult using the electrospinning method. The main aim of this study was developing a highly porous scaffold by poly (L-lactic acid) (PLLA)/chitosan blend using liquid-liquid phase separation (LLPS) technique, a fast and cost-benefit method, in order to use in nerve tissue engineering. In addition, the effect of different polymeric concentrations on morphology, mechanical properties, hydrophilicity, in vitro degradation rate and pH alteration of the scaffolds were evaluated. Moreover, cell attachment, cell viability and cell proliferation of scaffolds as candidates for nerve tissue engineering was investigated. PLLA/chitosan blend not only had desirable structural properties, porosity, hydrophilicity, mechanical properties, degradation rate and pH alteration but also provided a favorable environment for attachment, viability, and proliferation of human neuroblastoma cells, exhibiting significant potential for nerve tissue engineering applications. However, the polymeric concentration in blend fabrication had influence on both characteristics and cell responses. It concluded that PLLA/chitosan nanofibrous 3-D scaffold fabricated by LLPS method as a suitable candidate for nerve tissue engineering.

Recent developments in antibody derivatives against colorectal cancer; A review.

Colorectal cancer (CRC) is the fourth most common cause of cancer and mortality worldwide and is the third most common cancer in men and women. Surgery, radiotherapy, and chemotherapy are conventionally used for the treatment of colorectal cancer. However, these methods are associated with various side effects on normal cells. Thus, new studies are moving towards more effective and non-invasive methods for treatment of colorectal cancer. Targeted therapy of CRC is a promising new approach to enhance the efficiency and decrease the toxicity of the treatment. In targeted therapy of CRC, antibody fragments can directly inhibit tumor cell growth and proliferation. They also can act as an ideal carrier for targeted delivery of anticancer drugs. In the present study, the structure and function of different formats of antibody fragments, immune-targeted therapy of CRC using antibody fragments will be discussed.

Elevated homocysteine, as a biomarker of cardiac injury, in panic disorder patients due to oxidative stress.

BACKGROUND AND OBJECTIVE: Patients with panic disorder (PD) suffer from elevated oxidative stress as a consequence of serotonin metabolism disorder. These patients have elevated serotonin concentration and catabolism of serotonin via monoamine oxidase. The aim of the present study was to evaluate serum homocysteine concentration and its relationship with oxidative stress level in PD patients, regarding homocysteine as a diagnostic biomarker of heart disease. MATERIALS & METHOD: Sixty patients with PD according to the DSM-5 diagnostic criteria for a panic attack and 60 healthy individuals were included in the present study. Peripheral venous blood samples were taken from patients. Erythrocytes and serum were separated from blood, and RBC hemolysates were prepared to investigate oxidative stress indices including glutathione and glutathione peroxidase. Serum homocysteine and carbonyl groups concentrations were measured in all samples. Data were analyzed using ANOVA, and p < .05 was considered significant. RESULTS: Results showed that serum carbonyl groups concentration was significantly higher in patients with PD than in healthy individuals (p < .001). The results also indicated decreased serum glutathione concentration and glutathione peroxidase activity in patients (p < .003). In addition, elevated homocysteine concentration in PD patients serum was observed during the present study (p < .003). CONCLUSION: Our findings support that patients with PD experience higher levels of oxidative stress, due to impaired serotonin metabolism, which is related to the prognosis of heart disease in these patients.

Changes in mean platelet volume and hematologic indices in patients with panic disorder due to oxidative stress.

OBJECTIVE: Cardiovascular disorders are common in patients with panic disorder (PD), usually mediated by platelets. The present study was conducted to evaluate oxidative stress conditions and complete analysis of blood cells in patients with PD. SETTING AND SAMPLE POPULATION: Sixty healthy individuals and 60 patients were included in the study. Whole blood and serum samples were obtained from patients and controls. MATERIALS & METHOD: Hematological studies, including blood cells count, hemoglobin, and hematocrit, were carried out on whole blood samples. In addition, oxidative stress indices including total antioxidant capacity, free oxygen species, and malondialdehyde concentration were measured in serum samples. RESULTS: Results showed that patients with PD had a significant increase in mean platelet volume index (MPV), platelet distribution width (PDW), red blood cell distribution width (RDW), and mean corpuscular hemoglobin concentration (MCHC) compared with healthy subjects (p < .05). Also, oxidative stress indices were significantly elevated in patients with PD compared with control group (p < .05). CONCLUSION: Elevated MPV is a hematologic indicator for patients with PD. This disorder may be caused by impaired serotonin metabolism, resulting in increased oxidative stress, as well as in platelet serotonin transporters. Regarding elevated oxidative stress, the risk of cardiovascular complications is high in patients with PD.

The importance of long non-coding RNAs in neuropsychiatric disorders.

In the last decade, transcriptome analyses have discovered thousands of long non-coding RNAs (lncRNAs) which are assumed as a fundamental part of the gene regulatory networks in the cell. Intriguingly, lncRNAs are abundantly enriched in the brain, displaying elaborate spatiotemporal expression profiles and modulation. They diversely participate in the delicate regulation of the central nervous system (CNS) development including self-renewal maintenance, cell fate decision, synapse plasticity, synaptogenesis and memory formation. Moreover, lncRNAs have vastly demonstrated correlations with mental illnesses such as neuropsychiatric disorders (NPDs), implying the vital jobs of these yet poorly-understood transcripts. Here, we underlie the accumulating evidence for the significance of lncRNAs in neural networks and their impairment in several NPDs including autism spectrum disorder (ASD), schizophrenia (SZ), intellectual disability (ID), major depressive disorder (MDD), Rett syndrome (RTT) and others.

Serum DKK1 is correlated with γ peak of serum electrophoresis in multiple myeloma: a multicenter biomarker study.

Aim: DKK1 is reported to be produced at high levels by myeloma cells. Therefore, the applicability of DKK1 as a tumor marker for multiple myeloma (MM) diagnosis was examined. Methods: Serum samples were collected and analyzed by DKK1 concentration kit and capillary zone electrophoresis. Then, the obtained results were statically analyzed. Results: It has been determined that the 10 ng/ml of DKK1 is the optimal level for MM diagnosis. Moreover, there was an ascending linear correlation between the DKK1 concentration and γ peak. Discussion: The observed correlation could be rooted in the positive feedback loop between MM cells and the mesenchymal stem cells. In view of these results, DKK1 could be deemed as diagnostic marker for MM.

Saffron carotenoids (crocin and crocetin) binding to human serum albumin as investigated by different spectroscopic methods and molecular docking.

Therapeutic effects of saffron ingredients were studied in some diseases. The pharmacokinetics and pharmacodynamics of these ingredients were also studied, but their transport mechanism is not clearly known. Serum albumin has been known as the most important transporter of many drugs in the body that affects their disposition, transportation, and bioavailability. Here, we investigated the interaction of crocin (Cro) with HSA, for the first time, and compared with the crocetin (Crt)-HSA interaction. UV and fluorescence spectroscopy, circular dichroism (CD), and molecular docking was applied to investigate the possibility and mechanism of binding of HSA with these natural carotenoids. The gradually addition of Cro increased HSA absorbency at 278 nm, while Crt decreased it. Both of these changes induced HSA unfolding that was confirmed by the decreased α-helix content, as determined by the CD. Both carotenoids quenched HSA fluorescence emission, but with different mechanisms. The Stern-Volmer plots indicated a dynamic quenching of intrinsic emission of HSA due to Cro addition, while Crt quenching followed both static and dynamic quenching mechanisms. Docking results indicated binding of Cro/Crt in sub-domain IIA, Sudlow site I of HSA, which accompanied with the hydrogen bonding of Cro/Crt with Tyr138. The interaction of these ligands (Cro/Crt) caused HSA unfolding and affects the hydrophobic environment of Trp241, which result in the quenching of Trp fluorescence. The UV spectroscopy and fluorescence quenching data indicated the differences in the mechanisms of interaction of Cro/Crt with HSA, which is due to the differences in the structure and hydrophobicity of these ligands.

Inhibition of AGS Cancer Cell Proliferation following siRNA-Mediated Downregulation of VEGFR2.

OBJECTIVE: Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles in angiogenesis of different developmental mechanisms such as wound healing, embryogenesis and diseases, including different types of cancer. VEGFR2 is associated with cell proliferation, migration, and vascular permeability of endothelial cells. Blocking VEGF and its receptors is suggested as a therapeutic approach to prevent tumor growth. In this study, we aim to block VEGF signaling via small interfering RNA (siRNA) inhibition of VEGFR2. MATERIALS AND METHODS: In this experimental study, we used the RNA interference (RNAi) mechanism to suppress expression of the VEGFR2 gene. We conducted the 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), Western blot, and flow cytometry analyses of VEGFR2 expression. RESULTS: Real-time PCR and Western blot results showed that VEGFR2 expression significantly downregulated. This suppression was followed by inhibition of cell prolifera- tion, reduction of viability, and induction of apoptosis in the cancer cells. CONCLUSION: These findings suggest that VEGFR2 has a role in cell proliferation and tumor growth. Accordingly, it is suggested that VEGFR2 can be a therapeutic target for controlling tumor growth and proliferation.

Oxidative stress and anti-oxidant defense system in Iranian women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common disorder of infertility which affects more than 100 million women. It is characterized by chronic anovulation, hyper androgenism and obesity. PCOS is also associated with oxidative stress changes. OBJECTIVE: Here, we aimed to investigate the level of antioxidants and oxidative stress in Iranian women with PCOS as a predictive factor for cardiovascular disease for the first time in Iran. MATERIALS AND METHODS: In this cross sectional study 30 women with PCOS and 30 healthy women were included. C-reactive protein, serum insulin, advanced oxidation protein products, and level of total antioxidants status were measured from blood samples. RESULTS: The levels of serum insulin, C-reactive protein, advanced oxidation protein productswere significantly increased in women with PCOS compared with healthy women but there was a decrease in level of total antioxidants status in PCOS women. CONCLUSION: These changes show that oxidative stress contributes to PCOS and the decrease of antioxidants leads to increase of oxidation products contributing to PCOS.