Designing a novel fusion protein from Streptococcus agalactiae with apoptosis induction effects on cervical cancer cells.

Cervical cancer remains life-threatening cancer in women around the world. Due to the limitations of conventional treatment approaches, there is an urgent need to develop novel and more efficient strategies against cervical cancer. Therefore, the researchers attend to the alternative anti-cancer compounds like bacterial products. Rib and α are known as surface proteins of Streptococcus agalactiae with immunologic effects. In the present study, we designed a new anti-cancer fusion protein (Rib-α) originating from S. agalactiae with in silico methods, and then, the recombinant gene was cloned in the pET-22 (+) expression vector. The recombinant protein was expressed in E. coli BL21. To purify the expressed protein, we applied the Ni-NTA column. The molecular mechanism by which Rib-α is cytotoxic to cancer cells has been discussed based on MTT, flow cytometry, and real-time PCR methods. The engineered fusion protein suppressed the proliferation of the cancer cells at 180 µg/ml. Cytotoxic assessment and morphological changes, augmentation of apoptotic-related genes, upregulation of caspase-3 mRNA, and flow cytometric analysis confirmed that apoptosis might be the principal mechanism of cell death. According to our findings, Rib-α fusion protein motivated the intrinsic apoptosis pathway. Therefore, it can be an exciting candidate to discover a new class of antineoplastic agents.

The correlation of long non-coding RNAs IFNG-AS1 and ZEB2-AS1 with IFN-γ and ZEB-2 expression in PBMCs and clinical features of patients with coronary artery disease.

BACKGROUND: Aberrant expression of long non-coding RNAs (lncRNAs) can contribute to the pathogenesis of coronary artery disease (CAD). In this study, we aimed to evaluate the expression of lncRNA interferon γ-antisense 1 (IFNG-AS1), zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1), and their direct target genes (IFN-γ and ZEB2, respectively) in peripheral blood mononuclear cell (PBMC) from CAD and healthy individuals. METHODS AND RESULTS: We recruited 40 CAD patients and 40 healthy individuals. After doing some bioinformatics analyses, the expressions of IFNG-AS1/ ZEB2-AS1 lncRNAs and IFN-γ/ ZEB2 in PBMCs were measured using quantitative real-time PCR. The possible correlation between the putative lncRNAs and disease severity was also assessed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive role of lncRNAs as diagnostic biomarkers in CAD patients. The expressions of IFNG-AS1 lncRNA as well as IFN-γ and ZEB2 genes were significantly reduced in CAD patients compared to healthy subjects. In contrast, the expression of ZEB2-AS1 was up-regulated in these patients. Linear regression analysis unveiled that there is a positive correlation between the expression of IFNG-AS1 and IFN-γ, also similarly, ZEB2-AS1 and ZEB2 in PBMCs of subjects. Moreover, the expression of IFNG-AS1 and ZEB2-AS1 correlated with the Gensini score. The area under the ROC curves ranged from 0.633-0.742 for ZEB2-AS1/ZEB2 and IFNG-AS1/IFN-γ, respectively. CONCLUSIONS: Our results indicated that the dysregulation of IFNG-AS1/IFN-γ and ZEB2-AS1/ZEB2 in PBMCs of CAD patients may be involved in CAD pathogenesis.

Responses of platelet CD markers and indices to resistance exercise with and without blood flow restriction in patients with type 2 diabetes.

BACKGROUND: Diabetes mellitus is a common disorder with the risk of vascular injury. OBJECTIVE: The aim of this study was to compare the effects of low-intensity resistance exercise with blood flow restriction versus high-intensity resistance exercise on platelet CD markers and indices in patients with type 2 diabetes. METHODS: Fifteen female patients with type 2 diabetes (Mean±SD; age, 47.6±7.2 yrs) randomly completed two resistance exercise at an intensity corresponding to 20% and 80% of one-repetition maximum (1-RM), with and without blood flow restriction (REBFR and RE), respectively. We measured markers of platelet activation (P-selectin, GpIIb/IIIa, and CD42) and platelet indices before and immediately after exercise, and after 30 min recovery. RESULTS: Platelet count (PLT) and plateletcrit (PCT) increased in response to REBFR more than the RE (p < 0.05), though, no significant differences in PDW and MPV were observed (p < 0.05). Although P-selectin (CD62P), CD61, CD41, and CD42 were reduced following resistance exercise in both trials, these reductions were non-significant (p < 0.05). Besides, no significant between-group differences were found for platelet CD markers (p < 0.05). CONCLUSIONS: It is concluded that REBFR induces thrombocytosis, but responses of platelet CD markers in patients with type 2 diabetes are similar following low-intensity REBFR and high-intensity RE.

Anti-angiogenic peptides application in cancer therapy; a review.

Cancer is a disease advanced via surplus angiogenesis. The development of new anti-angiogenic therapeutic agents with more efficacy and fewer side effects is still quite necessary. Conventional therapies saving the life of many cancer patients but due to drug resistance and lack of specificity utilizing these methods is faced with limits. Recently, new therapeutic agents have been developed and used to treat cancers such as scaffold proteins, monoclonal antibodies, tyrosine kinase inhibitors, and peptides. In antiangiogenic drug development, anti-angiogenic peptides design is a significant aim. Peptides have developed as substantial therapeutics that are being carefully investigated in angiogenesis-dependent diseases because of their high penetrating rate into the cancer cells, high specificity, and low toxicity. In this review, we focus on anti-angiogenic peptides in the field of cancer therapy that are designed, screened, or derived from nanobodies, mimotopes, phage displays, and natural resources.

Isolation and characterization of halophilic bacteria with the ability of heavy metal bioremediation and nanoparticle synthesis from Khara salt lake in Iran.

Increasing environmental pollutants such as heavy metals have become one of the most severe health dangers because of rapid industrialization. Exposure to lead and nickel heavy toxic metals can lead to hazardous diseases affecting most of the organs in humans. Bioremediation is a process that uses the ability of microorganisms or plants to detoxify environmental contaminants at lower costs than physicochemical techniques. This study isolated halophilic bacteria from Khara salt lake in Iran and screened their ability to resist lead and nickel. After screening stages, three selected strains including Bacillus sp. A21, Oceanobacillus sp. A22 and Salinicoccus A43 were identified by16S rDNA sequencing and the related sequences were submitted to GeneBank with accession IDs MN588312, MN588313, and MN 588,314, respectively. These strains resist 7.2 mM, 4.1 mM, and 6.7 mM lead and 3.6 mM, 3.7 mM, and 4.1 mM nickel, respectively. Investigation of growth pattern and evaluation of bioremediation ability by atomic absorption spectroscopy revealed that Bacillus sp. A21 could decrease lead and nickel in culture medium up to 97.5% and 76%, respectively. Oceanobacillus sp. A22 showed higher lead bioremediation rate (98.8%) and lower nickel-bioremediation rate (73.5%). Salinicoccus sp. A43 showed the least bioremediation ability (92% lead and 71.7% nickel). The ability of selected strains to synthesize lead and nickel nanoparticles was evaluated using UV/Vis spectrophotometry and Energy-Dispersive X-ray Spectroscopy (EDX). Particle dimensions were measured using Scanning Electron Microscopy (SEM). Bacillus sp. A21 and Oceanobacillus sp. A22 strains were able to synthesize lead nanoparticles; however, Salinicoccus sp. A43 could synthesize both lead and nickel nanoparticles.

Designing and Development of a Tandem Bivalent Nanobody against VEGF165.

BACKGROUND: Inhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody. METHODS: Homology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively. RESULTS: RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein. CONCLUSION: Our data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

Evaluation of antibacterial activity of enterocin A-colicin E1 fusion peptide.

OBJECTIVES: Bacterial resistance to most common antibiotics is a harbinger of the requirement to find novel anti-infective, antimicrobials agents, and increase innovative strategies to struggle them. Numerous bacteria produce small peptides with antimicrobial activities called bacteriocin. This study aimed to investigate the antibacterial properties of the fusion protein of Enterocin A and Colicin E1 modified against pathogens. MATERIALS AND METHODS: Analysis of recombinant bacteriocin Enterocin A and Colicin E1 (ent A-col E1) was performed to assay the stability and antibacterial activity of this fusion protein. The pET-22b vector was employed to express the coding sequence of the ent A-col E1 peptide in Escherichia coli BL21 (DE3). Minimum inhibitory concentration (MIC), disk diffusion, and time-kill tests were performed to evaluate the antibacterial activity of the ent A-col E1 against Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 10536), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 33591). RESULTS: The suggested recombinant peptide had good antibacterial activity against both Gram-negative and Gram-positive pathogens. It has also good stability at various temperatures, pH levels, and salt concentrations. CONCLUSION: Because bacteriocins are harmless compounds, they can be recommended as therapeutic or preventive supplements to control pathogens. According to the obtained results, the ent A-col E1 peptide can serve as an efficient antibacterial compound to treat or prevent bacterial infections.

New Proline, Alanine, Serine Repeat Sequence for Pharmacokinetic Enhancement of Anti-VEGF Single-Domain Antibody.

Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.

Effective removal of organic pollution by using sonochemical prepared LaFeO3 perovskite under visible light.

In the present work, LaFeO3 perovskite was prepared via ultrasonic probe with power of 60 W and frequency of 18 KHz. LaFeO3 nanorods were formed when sonication time was 20 min. In this research, green materials including corn, starch, and rice were used to control the size, morphology, and purity of final products. As-prepared LaFeO3 nanostructures were used to purify water containing organic contaminants. LaFeO3 nanostructures prepared by using corn, starch, and rice showed higher photocatalytic activity compare to LaFeO3 nanostructures without natural capping agents. Using corn increased degradation efficiency by 65% under visible light. XRD results show that Fe2O3 appeared as an impurity when starch was used to prepare LaFeO3 nanostructures. This impurity significantly boosts the degradation efficiency under UV light. Fe2O3 under UV light act as co-absorbent and boost efficiency by 43%. LaFeO3 nanostructures were characterized by XRD, EDX, SEM, CV, BET, TEM, DRS and FT-IR.

The effect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have accelerated atherosclerosis as a pro thrombotic state that is associated with the platelet activation priming. Platelets, which undergo the continuous mild stimulation, may lose their sensitivity to react to a strong stimulation. The present study aimed to investigate activation responses of platelets to mild and subsequent strong stimulations in patients with T2DM and healthy individuals. METHODS: Blood samples, which were taken from 40 patients with T2DM and 35 healthy individuals, were collected into the citrate containing tubes. The samples were subjected to the soft centrifugation to prepare the platelet rich plasma (PRP). Platelets in PRP samples were treated at a low (1 µM) concentration and then at a high (10 µM) concentration of ADP. Before and after stimulation with different doses of ADP, levels of CD62P expression and formation of platelet micro particles (PMPs) were measured using a flow cytometry method. RESULTS: The platelets from patients with T2DM had higher levels of CD62P expression before any stimulation (P = 0.003) than control samples. Platelets, which underwent the mild stimulation, indicated lower responses to CD62P expression, but higher PMPs formation after stimulation with high dose of ADP. Patients with T2DM had higher platelet micro particles in all states with the ADP stimulation. (P = 0.004, SD: ±74.52). CONCLUSIONS: The flow cytometry data indicated that platelets were pre-active and associated with metabolic conditions in patients with type 2 diabetes mellitus. The induction of desensitization state helped platelets to reduce the platelet activation and sensitivity to ADP in a diabetic environment. Furthermore, the production of platelets micro-particles was high in the patients; and desensitized platelets were more susceptible to shedding of micro-particles.

Differential Expression of HSP90ß in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin.

BACKGROUND: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including HSP90α and HSP90ß in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. MATERIAL AND METHODS: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. Immunofluorescence and western blotting techniques were used to determine the expression of HSP90ß in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of HSP90α. RESULTS: The MTT assay results showed that the MDA-MB- 231 cells (IC50=14.521 µM) were more sensitive than the MCF-7 cells (IC50=16.3315 µM) to DOX. The immunofluorescence results indicated that the expression of HSP90α in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that HSP90ß expression decreased in the MCF-7 cells but increased in the MDA-MB- 231 cells after DOX treatment. Conclusion: The obtained results suggested that HSP90α and HSP90ß expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, HSP90α expression was reduced while HSP90ß was found to be overexpressed following DOX treatment.

The Radioprotective Effects of Curcumin and Trehalose Against Genetic Damage Caused By I-131.

Background: Thyroid cancer has been growing rapidly during the last decades. Radioiodine-131 (I-131) as an appropriate therapy modality is currently using in the treatment of cancer and hyperthyroidism diseases. This radiotracer is considered as a cause of oxidative DNA damage in nontarget cells and tissues. The aim of this study was to investigate the effects of curcumin and trehalose on the level of DNA double-strand breaks (DSBs) caused by I-131 in human lymphocytes. Materials and Methods: First, 6-mL blood samples were taken from each of the five volunteers. After 1 h of preincubation with the antioxidants, a total of 20 µCi I-131/2 mL (blood + NaCl) was added to each sample, and then, the samples were reincubated for 1 h. Lymphocytes were separated and the mean DSB levels were measured for each sample through γ-H2AX assay to evaluate the effects of antioxidants. Results: After 1-h incubation with I-131, the DSBs increased by 102.9% compared to the control group (0.343 vs. 0.169 DSB/cell; P = 0.00). Furthermore, compared to the control + I-131 group, curcumin and trehalose reduced the DSBs by 42% and 38%, respectively. There was a significant decrement (P = 0.00) in the levels of DSBs of the curcumin + I-131 and trehalose + I-131 subgroups compared to the control + I-131 subgroup. Furthermore, there was no significant relationship between the radioprotective effect of curcumin and trehalose (P = 0.95). Conclusion: The use of curcumin and trehalose as antioxidant can reduce the numbers of DSBs caused by I-131. Meanwhile, the radioprotective effect of curcumin was more than trehalose.

Vitamins E and C Prevent DNA Double-strand Breaks in Peripheral Lymphocytes Exposed to Radiations from Iodine-131.

PURPOSE: Iodine-131 is used as a radiopharmaceutical to treat thyroid cancer. The current study aimed to evaluate the effects of vitamins E and C on the level of DNA double-strand breaks (DSBs) caused by Radioiodine-131 (I-131) in human lymphocytes. MATERIALS AND METHODS: Whole blood samples from human volunteers were incubated with a certain concentration of vitamins. After 1-h incubation, the samples were incubated with 20 µCi I-131/2 mL (blood + NaCl) for 1 h. To evaluate the effects of antioxidants, lymphocytes were separated, and the mean DSBs/cell was measured for each sample through γ-H2AX assay. RESULTS: After 1-h incubation with 20 µCi I-131/2 mL (blood + NaCl), iodine-131 increased the level of DSBs by 102.9%, compared with the background group. Vitamins E and C reduced the level of DSBs by 21.5% and 36.4%, respectively. CONCLUSION: Using vitamins E and C as antioxidants can reduce the toxicity of I-131. Furthermore, vitamin C provided the more protection for DNA, compared with vitamin E.

Evaluation of Ameliorative Potential of Vitamins E and C on DNA Double Strand Break (DSB) in Patients Undergoing Computed Tomography (CT): A Clinical Study.

Computed tomography (CT) is one of the most important diagnostic X-ray procedures which plays an important role in increasing the patient dose values. The purpose of this clinical study was to evaluate the efficacy of vitamins E and C in lowering down the level of DNA double strand break (DSB) caused by CT scan. Sixty patients for abdomen/pelvic enhanced CT scan were randomly assigned to placebo (control), vitamin C, and vitamin E groups. The patient blood samples were taken before and immediately after the CT scan. Counting the number of DSB was performed using γ-H2AX method as a sensitive biomarker. Immediately after the CT scan, the mean number of DSBs/cell increased in all three groups of control (131%, P<0.001), vitamin C (103%, P <0.001), and vitamin E (66%, P<0.001) compared to their mean before the CT scan. Furthermore, the results showed that vitamin E decreased the mean number of DSBs/cell by 22% in comparison with the control group (P =0.023), whereas vitamin C had no significant effect on reducing the DSB (<3%, P =0.741). It is concluded that the administration of vitamin E one hour before the CT scan, significantly decreases DSB levels.

Platelet activation and function in response to high intensity interval exercise and moderate continuous exercise in CABG and PCI patients.

BACKGROUND: The effects of high intensity interval training (HIIT) on inflammatory markers and endothelial function have been extensively shown. However, the acute effect of HIIT on platelet activation and function in patients with recent revascularization is unclear. OBJECTIVE: The purpose of present study was to compare the responses of platelet activation (CD62P) and function (platelet aggregation) to high intensity interval exercise (HIIE) and moderate continuous exercise (MCE) in coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI) patients. METHODS: Thirty patients who had CABG or PCI were randomly divided into HIIE, MCE and control groups. After determining the VO2peak, subjects in the MCE group carried out 30 min of continuous exercise at 60% of VO2peak, whereas, the subjects in HIIE group performed an interval protocol consisted of 8 repetitions of 2 min activity (running on treadmill) at 90% of VO2peak interspersed by 2 min of active recovery between repetitions at 30% of VO2peak .  Subjects in control group were seated and had no activity for the same period of time. Two blood samples were collected before and immediately after exercise and were analyzed for markers of platelet activation and function. RESULTS: Data analyzes revealed that increases in platelet aggregation induced by ADP and corrected for increases in platelet count in response to MCE trial was significantly lower than HIIE group (P < 0.05). In addition, responses of CD62P to MCE trial was significantly lower compared to HIIE group (P < 0.05). Changes in plateletcrit and platelet distribution width were significantly different among the three trials where the PCT and PDW following the HIIE were higher than MCE. Platelet count increased significantly (P < 0.05) by 13% following HIIE trial. CONCLUSIONS: Based on the findings of the present study it could be concluded that the risk of exercise-induced thrombosis is higher during HIIE than MCE in patients with recent revascularization.

Measurement of Entrance Skin Dose and Calculation of Effective Dose for Common Diagnostic X-Ray Examinations in Kashan, Iran.

The knowledge of the radiation dose received by the patient during the radiological examination is essential to prevent risks of exposures. The aim of this work is to study patient doses for common diagnostic radiographic examinations in hospitals affiliated to Kashan University of Medical sciences, Iran. The results of this survey are compared with those published by some national and international values. Entrance surface dose (ESD) was measured based on the exposure parameters used for the actual examination and effective dose (ED) was calculated by use of conversion coefficients calculated by Monte Carlo methods. The mean entrance surface dose and effective dose for examinations of the chest (PA, Lat), abdomen (AP), pelvis (AP), lumbar spine (AP, Lat) and skull (AP, Lat) are 0.37, 0.99, 2.01, 1.76, 2.18, 5.36, 1.39 and 1.01 mGy, and 0.04, 0.1, 0.28, 0,28, 0.23, 0.13, 0.01 and 0.01 mSv, respectively. The ESDs and EDs reported in this study, except for examinations of the chest, are generally lower than comparable reference dose values published in the literature. On the basis of the results obtained in this study can conclude that use of newer equipment and use of the proper radiological parameter can significantly reduce the absorbed dose. It is recommended that radiological parameter in chest examinations be revised.

Variations in Intraplatelet Phospho-VASP Expression Due to Pre-analytical Sample Preparations, Illustration of a Quality Control Issue in Platelet Pharmacology.

Intraplatelet vasodilator-stimulated phosphoprotein (VASP) analysis is a commonly used laboratory approach for monitoring of the anti-platelet therapy with adenosine diphosphate (ADP) receptor blocking agents; however, it's testing in clinical laboratory needs a high level of experience and proficiency. The ability to recognize how the pre-analytical variations can change the results would be helpful for the interpretation of data from intraplatelet VASP analysis. The aim of this study was to describe the possible differences of intraplatelet phospho-VASP expression between washed and platelet rich plasma (PRP) samples, both at baseline levels and following experimentally induction of VASP phosphorylation. PRP and washed platelet samples were treated with different inducers of VASP phosphorylation, including forskolin (10 µM), prostaglandin E1 (PGE1) (50 nM) and sodium nitro-prusside (SNP) (100 µM). Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine(157)-VASP or anti P-Serine(239)-VASP, the samples were subjected to flow cytometric analysis to monitor the levels of intraplatelet phospho-VASP expression. Washed platelet samples tend to show increased expression of intraplatelet P-Serine(157)-VASP at baseline state and also more expression of P-Serine(157)-VASP and P-Serine(239)-VASP in response to forskolin and SNP, compared with PRP samples. Though, reduced levels of PGE1-induced VASP phosphorylation at both residues were detected for washed platelets. In this study we have provided some background information required for performing of intraplatelet VASP analysis on differently handled platelet samples and interpretation of the obtained results.

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions.

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.

Modulation of hyperthermia-induced platelet aggregation inhibition in the presence of urea.

PURPOSE: This study has been conducted to evaluate the effect of urea on aggregation responses of heat-treated platelets. MATERIALS AND METHODS: The urea was added to platelet-rich plasma (PRP) samples in final concentrations of 50 and 100 mM. PRP samples, with or without exogenous urea, were incubated at 37 °C, 39 °C and 41 °C for 90 min and then were stimulated with adenosine diphosphate (ADP) or epinephrine for measuring of platelet aggregation responses. The average reduction in aggregability of heat-treated samples with reference to mean value obtained for control samples treated at 37 °C was expressed as inhibition percentage. RESULTS: Aggregation responses of the samples treated in the presence of 50 mM and 100 mM urea were significantly less inhibited by hyperthermia treatments compared with those treated without exogenous urea. CONCLUSION: The results indicate that the inhibitory effect of hyperthermia on platelet aggregation responses could be significantly modulated by urea.