Simultaneous Effects of High Intensity Interval Training and Human Amniotic Membrane Scaffold on Rat Tibialis Anterior Vascularization and Innervation after Volumetric Muscle Loss Injury.

BACKGROUND: Despite the high regenerative capacity of skeletal muscle, volumetric muscle loss (VML) is an irrecoverable injury. One therapeutic approach is the implantation of engineered biologic scaffolds. OBJECTIVE: To investigate the simultaneous effect of high intensity interval training (HIIT) and the use of decellularized human amniotic membrane (dHAM) scaffolds on vascularization, growth factor, and neurotrophic factor gene expression, and muscle force generation in the tibialis anterior (TA) of rats after VML injury. METHODS: VML injury was created in the TA of 24 rats, which were randomly divided into two groups-12 animals with and 12 without the use of a dHAM scaffold. After injury, each group was further divided into two groups of 6 animals each-sedentary and HIIT. Blood vessels were visualized and counted by hematoxylin and eosin staining. The PowerLab converter assay was used to evaluate isometric contraction force. The relative expression of neurotrophic factors and growth factor genes was measured with reverse transcription PCR (RT-PCR). RESULTS: The number of blood vessels in the whole regenerating areas showed a significant difference in the dHAM-HIIT and dHAM-sedentary groups compared to the sedentary group without dHAM (p=0.001 and p=0.003, respectively). BDNF and GDNF mRNA levels in the dHAM-HIIT group were significantly (p<0.05) higher than those in other groups; NGF mRNA levels did not differ significantly among groups. Isometric contraction force in the dHAM-HIIT group was significantly (p=0.001) greater compared to the sedentary group without dHAM. CONCLUSION: Combined use of dHAM scaffoldsand HIIT would improve the structure of the injured muscle during regeneration after VML by better vascular perfusion. HIIT leads to greater force generation and innervation by modulating neurotrophic factor synthesis in regenerating muscles.

Osteopontin siRNA does not confer resistance to toxic effects of parthenolide in Jurkat cells.

BACKGROUND: Osteopontin (OPN) plays a critical role in cell proliferation and drug resistance in cancer treatment and hematological malignancies. In T cell acute lymphoblastic leukemia, most initial therapies can induce remission while some patients then relapse and do not respond well to chemotherapy. The sesquiterpene lactone parthenolide (PTL) can induce apoptosis in a variety of cancer cell lines via inhibition of pro-inflammatory transcription factor nuclear factor kappa B and has anti-tumor activity in acute lymphoblastic leukemia treatment. AIM: To study the role of OPN in conferring in vitro resistance to PTL in Jurkat cells. METHODS: Jurkat cells were cultured with 8-20 µm PTL for 48 h. Transfection with OPN siRNA was provided. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI. Quantitative real-time polymerase chain reaction was used to measure OPN gene expression using the 2-2-ΔΔCt method. RESULTS: PTL has cytotoxic and apoptotic effect on Jurkat cells with IC50 values of 16.1 µm, and growth inhibition effect of PTL does not differ significantly in combination with OPN-siRNA. OPN gene expression is not affected by PTL. CONCLUSIONS: Parthenolide induces apoptosis in Jurkat cells, but inhibition of osteopontin gene expression with siRNA does not reduce apoptotic effect of parthenolide.

Parthenolide reduces gene transcription of prosurvival mediators in U937 cells.

In acute myeloid leukemia (AML) the functional abnormalities of osteopontin (OPN), NF-kB, PI3K/AKT/mTOR/PTEN pathway or ß-catenin have been considered. AIM: To analyze the response of U937 cells to parthenolide (PTL) through the involvement of expression of OPN protein, RelB, AKT1, mTOR, PTEN and ß-catenin genes. MATERIALS AND METHODS: The U937 cells were treated with PTL at concentrations of 4 µM (IC25) or 6 µM (IC50) and with OPN siRNA for MTT assay and colony forming assay. Western blot analysis using antibodies against OPN was performed with lysates of PTL-treated cells. Quantitative real-time polymerase chain reaction was performed using primers for OPN siRNA, RelB, AKT1, mTOR, PTEN and ß-catenin. RESULTS: PTL reduces OPN protein level and down-regulates RelB mRNA in U937 cell line. Suppression of OPN with siRNA increases the cytotoxic effects of PTL. Also, mRNA expression of AKT1, mTOR, PTEN, and ß-catenin decreases with PTL or OPN siRNA. CONCLUSION: Sensitivity of U937 cells to PTL can be associated with the reduction in expression of prosurvival mediators.

Aberrant promoter hypermethylation of selected apoptotic genes in childhood acute lymphoblastic leukemia among North Indian population.

Promoter hypermethylation mediates gene silencing in many neoplasms. Acute leukemia has been reported to harbor multiple genes aberrantly silenced by hypermethylation. AIM: In present study, we investigated the prevalence of hypermethylation of caspase-8 (CASP8), TMS1 and DAPK genes in correlation with clinicopathological factors in childhood acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: A case-control study has been conducted based on bone marrow and peripheral blood samples from 125 ALL patients and 100 sex-age matched healthy controls. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing PCR was performed to analyze the methylation status of these genes. Reverse transcription PCR and real time PCR was carried out to determine changes in the mRNA expression level of the genes due to hypermethylation. RESULTS: Hypermethylation of the 5´CpG islands of the CASP8, TMS1 and DAPK gene promoters was found in 3.2, 6.4, and 13.6% of 125 childhood ALL samples from north Indian population, respectively. There were significant differences in pattern of hypermethylation of TMS1 (p = 0.045) and DAPK (p < 0.001) between patients and healthy controls. Down-regulation of mRNA expression was found in cases in which CASP8, TMS1 and DAPK were hypermethylated. CONCLUSIONS: The present study indicated the impact of hypermethylation-mediated inactivation of CASP8, TMS1 and DAPK genes, which is associated with risk of childhood ALL. This abnormality occurs in leukemogenesis and it may be used as a biomarker and for predicting the prognosis of ALL.

A COMPARATIVE ANALYSIS OF METHYLATION STATUS OF TUMOR SUPPRESSOR GENES IN PAIRED BIOPSY AND SERUM SAMPLES FROM CERVICAL CANCER PATIENTS AMONG NORTH INDIAN POPULATION.

Tumor-specific genetic or epigenetic alterations have been detected in serum DNA in case of various types of cancers. In breast cancer, the detection of tumor suppressor gene hypermethylation has been reported in several body fluids. Promoter hypermethylation of some genes like MYOD1, CALCA, hTERT etc. has also been detected in serum samples from cervical cancer. The present study is the first report on the comparison of promoter hypermethylation of tumor suppressor genes likep14, p15, p16, p21, p27, p57, p53, p73, RARß2, FHIT, DAPK, STAT1 and-RB1 genes in paired biopsy and serum samples from cervical cancer patients among north Indian population. This is also the first report on the hypermethylation of these genes in serum samples from cervical cancer patients among north Indian population. According to the results of the present study, promoter hypermethylation of these genes can also be detected in serum samples of cervical cancer patients. The sensitivity of detection of promoter hypermethylation in serum samples of cervical cancer patients as compared to paired biopsy samples was found to be around 83.3%. It was observed that promoter hypermethylation was mainly observed in the serum samples in the higher stages and very rarely in the lower stages. The present study clearly showed that serum of patients with cervical cancer can also be used to study methylated genes as biomarkers.

Computational design of an enantioselective molecular imprinted polymer for the solid phase extraction of S-warfarin from plasma.

An enantioselective molecular imprinted polymer for S-warfarin was designed computationally by using the density functional theory (DFT) at B3LYP/631G+ (d, p) level and Gaussian 2003 package. The effect of polymerization solvent was also evaluated by the polarizable continuum model (PCM) and it was based on the measurement of interaction energies (ΔE) between S-warfarin and monomers in different polymerization solvents. The computational method showed that the methacrylic acid (MAA) and acetonitrile (AN) had the highest stabilization energy for the pre-polymerization adducts. Additionally, the mole ratio of 1:3 give the highest ΔE, therefore, the polymer was synthesized by the thermal bulk polymerization method with the mole ratio of S-warfarin-(MAA)3. The enantioselective extraction of MIP for R and S-warfarin was evaluated by chiral separation chromatography and polarimetry methods. The results revealed that the proposed S-warfarin molecular imprinted polymer has a moderate recognition for extraction of R-warfarin in a racemic mixture and had no recognition for other foreign drugs. In a racemic mixture of R and S-warfarin, the polymer is able to remove about 20% of R-warfarin. The linearity between responses (peak areas) and concentrations of S-warfarin in plasma sample was found in the range of 15.4-3080ngmL(-1) (R(2)=0.999).The linear range for a racemic mixture of R, S-warfarin in plasma which has been obtained by RP-C18-HPLC-UV method, was 12.0-2500ngmL(-1) (R(2)=0.998). The polymer was used for analysis of a real sample and as expected the accurate results were obtained.

Chemical composition and antiprolifrative activity of Artemisia persica Boiss. and Artemisia turcomanica Gand. essential oils.

Essential oils obtained from aerial parts of Artemisia persica and Artemisia turcomanica were analyzed by GC/MS. While 28 components representing 91.01 % of A. persica were identified, the identity of 50 components, constituting 81.93 % of the total oil, was confirmed in A. turcomanica. ß-thujone was the main compound (75.23%) in A. persica while the major identified phytochemicals in A. turcomanica were 1,8-cineol (19.23%), camphor (15.55%) and filifolone (15.53%). Both of the essential oils were predominantly made up of monoterpenes. Time- and dose-dependent cytotoxic effects of A. persica and A. turcomanica on MCF-7 cell line evaluated by MTT assay at 24, 48 and 72 h, showed that the highest cytotoxic effect of A. persica and A. turcomanica were appeared at 72 h incubation. At that incubation period, CI50 of A. persica was found to be 0.15 µg/ml, while that of A. turcomanica was 0.1 µg/ml. Thus, cytotoxicity of A. turcomanica was slightly higher than A. persica which could be attributed to the higher content of sesquiterpene present in A. turcomanica. As a conclusion, these volatile oils could have chemotherapeutic potentials.

Hemospermia: long-term outcome in 165 patients.

Long-term course of hemospermia has not been addressed in the sexual medicine literature. We report our 15 years' experience. From 1997 to 2012, 165 patients presented with hemospermia. Mean age was 38 years. Mean follow-up was 83 months. Laboratory evaluation and testis and transabdominal ultrasonography was done in all. Since 2008, all sonographies were done by the first author. One patient had urinary tuberculosis, one had bladder tumor and three had benign lesions at verumontanum. One patient had bilateral partial ejaculatory duct obstruction by stones. All six patients had persistent, frequently recurring or high-volume hemospermia. All pathologies were found in young patients. In the remaining 159 patients (96%), empiric treatment was given with a fluoroquinolone (Ciprofloxacin) plus an nonsteroidal anti-inflammatory drug (Celecoxib). In our 15 years of follow-up, no patient later developed life-threatening disease. Diagnostic evaluation of hemospermia is not worthwhile in the absolute majority of cases. Advanced age makes no difference. Only high-risk patients need to be evaluated. The vast majority of cases may be safely and effectively treated with empiric therapy. Almost all patients do well in long term.

Chemical composition and anxiolytic evaluation of Achillea Wilhelmsii C. Koch essential oil in rat.

Herbal based remedies are used worldwide to treat psychiatric disorders. The aim of this study was to analyse the essential oil composition of Achillea Wilhemsii C. Koch (Asteraceae) and to evaluate its anxiolytic effects in the elevated plus maze (EPM) model of anxiety in rat. Gas chromatography/mass spectrometry (GC/MS) analysis of the essential oil showed that the main compounds of the oil were p-ocimen (23%), 1, 8-cineole (20.8%) and carvone (19.13%). The EPM results showed that 1 mg/kg (i.p.) of the oil significantly (P<0.05) increased the percentage of the time spent and the number of entries in the open arms of the maze while it did not change the total number of entries in the maze arms. These effects were not reversed with 2 mg/kg flumazenil and 5 mg/kg naloxone. We concluded that a minimum dose of 1 mg/kg of the oil has anxiolytic effects which are not probably mediated through GABA and opioid receptors.

Production of 13N by 12C(d,n)13N reaction in a medium energy plasma focus.

This paper explores the production of (13)N by bombardment of a carbon target by high energy deuterons in a medium energy plasma focus. A set of experiments in the energy range of 2.7-3.1kJ and initial pressure of 200-700Pa, with three or five shots in each experiment, was performed. A HPGe detector was used for gamma spectroscopy, and 511keV photons emitted by positron annihilation were utilized to measure the (13)N radioactivity. The highest activity of (13)N in these experiments was 14Bq which was acquired after five shots at a pressure of 450Pa and a 3.1kJ stored energy. Calculations based on thick target yield showed that at least 1.9×10(9) deuterons with energies higher than 330keV were ejected from the pinch region.

Polymorphisms of MTHFR and MTR genes are not related to susceptibility to childhood ALL in North India.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most worldwide common type of childhood cancer. Methylenetetrahydrofolate reductase (MTHFR) and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) participate in folate pathways and are known as critical factors for DNA integrity as well as DNA hypomethylation. The aim of this work is to investigate frequency of MTHFR (677C→T and 1298A→C) and MTR (2756A→G) polymorphisms and their interaction with respect to possible effect on risk of childhood ALL among North Indian population. PROCEDURE: A case control study from has been conducted on bone marrow and peripheral blood samples from 125 ALL patients and 100 sex-age matched healthy controls using PCR-RFLP method. RESULTS: No statistically significant differences were observed for different genotypes between patients and controls (p>0.05). Significant difference for the risk of ALL in individuals having genotype of MTHFR 677TT (OR=0.61, 95% CI=0.21-1.77) and MTHFR 1298CC (OR=0.56, 95% CI=0.18-1.68) was not observed. The correlation of SNP of MTR gene and risk of ALL was not observed, too. CONCLUSIONS: The differences in distribution of possible combined genotypes of MTHFR (677C→T, 1298A→C) and MTR (2756A→G) between ALL patients and controls were statistically insignificant.

Reversal of hypermethylation and reactivation of the RARß2 gene by natural compounds in cervical cancer cell lines.

Reactivation of tumour suppressor genes that have been silenced by promoter methylation is a very attractive molecular target for cancer therapy. The treatment of a squamous cervical cancer cell line, SiHa, with 20 µM curcumin and genistein resulted in demethylation of promoter of the RARß2 gene and led to the reactivation of the gene. The degree of methylation as observed by MSP decreased as the time period of treatment was increased from 72 h to 6 days. In HeLa cells (an adenocarcinoma cervical cancer cell line) there was also reversal of hypermethylation of the RARß2 gene after six days of treatment with 20 µM curcumin. However, allyl sulphide treatment (20 µM) did not cause the reversal of hypermethylation until 72 h of treatment in the SiHa cell line. This is the first report to show the reversal of hypermethylation of the RARß2 gene by genistein and curcumin in cervical cancer cell lines. Furthermore, these compounds acted as doublepronged agents as they caused apoptosis in the treated cervical cancer cell lines in addition to reversal of promoter hypermethylation.

Molecular fingerprinting of meticillin-resistant Staphylococcus aureus strains isolated from patients and staff of two Iranian hospitals.

Meticillin-resistant Staphylococcus aureus (MRSA) is an important cause of hospital-acquired infection. Methods for typing and epidemiological investigation of MRSA isolates have an important impact in detection of MRSA strains, source, transmission and control of these micro-organisms. The aims of this study were to study molecular diversity of MRSA isolates by randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), the surveillance efficacy of this method and determination of antibiotic resistance patterns of MRSA isolates. MRSA isolates were collected from clinical specimens and noses of 460 staff and inpatients admitted to Imam Khomeini and Paediatric Hospitals during a six-month period (2004-2005). Eighty MRSA strains, in which the presence of mecA gene had been confirmed by PCR, were subjected to RAPD-PCR using five primers and the results were summarised in a dendrogram to show the relationships between the test isolates. Antibiotic resistance patterns of MRSA isolates were also determined by disc agar diffusion method using 13 antibiotic discs according to Clinical and Laboratory Standards Institute guidelines. Forty-three RAPD-PCR profiles were detected. The test isolates were clustered into 18 taxa with 50% similarity, indicating the heterogeneity of our test isolates. MRSA isolates fell into 41 antibiotic resistance patterns. There was correlation between antibiotic resistance patterns and results of RAPD-PCR. Most of the MRSA isolates were multi-resistant.

Suppression of presynaptic responses to adenosine by activation of NMDA receptors.

The interactions between adenosine and NMDA receptors has been investigated using the paired-pulse paradigm in hippocampal slices. This technique allows the study of drug effects specifically at presynaptic terminals. The inhibitory effect of adenosine on population spikes, and the decrease of paired-pulse inhibition assessed using either population spikes or population excitatory postsynaptic potentials, were suppressed by performing the experiments in magnesium-free medium, or by superfusion of the slices with N-methyl-D-aspartate (NMDA) at a concentration (4 microM) which did not itself affect potential size. The suppressant effect of NMDA was prevented by 2-amino-5-phosphonopentanoic acid. All these interactions were still seen in the presence of bicuculline methobromide, 30 microM. Neither alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) nor kainate produced a suppression of adenosine responses. The presence of NMDA did not modify the effects of baclofen on population potentials or paired-pulse inhibition. Activating NMDA receptors by the induction of long-term potentiation or by superfusion with glycine also reduced significantly the effects of adenosine on population spikes and paired-pulse interactions. Increasing population potential size by a mechanism which did not involve the activation of NMDA receptors (increasing stimulus strength) did not change sensitivity to adenosine. When adenosine receptor-selective agonists were tested, it was found that NMDA did not modify the inhibitory effect of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine, but did enhance the excitatory effect of the adenosine A(2A) receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). The combined response to NMDA and CGS21680 was prevented by the adenosine A(2A) receptor selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). It is concluded that NMDA receptor activation can suppress neuronal sensitivity to adenosine by acting at presynaptic sites, and that this interaction results from an increase in the excitatory action of adenosine A(2A) receptors, rather than a depression of A(1) receptor function.

Presynaptic P2 receptors?

Although the emphasis in ATP research has been on postjunctional receptors, there is also evidence for presynaptic receptors regulating transmitter release in the autonomic nervous system. Recent work has attempted to identify similar mechanisms in the central nervous system. Some of the existing results can be explained by the metabolism of nucleotides to adenosine or adenosine 5'-monophosphate (AMP). However, studies of presynaptic effects using sensitive electrophysiological tests such as paired-pulse interactions indicate that nucleotides can act at presynaptic sites, but that their effects may be mediated by a release of adenosine. Results are also described which indicate that, under some conditions, nucleotides can mediate phenomena such as long-term potentiation, which probably involves a significant presynaptic element. In part these effects may involve a nucleotide-induced release of adenosine and the simultaneous activation of P1 and P2 receptors.

Complex hippocampal responses to ATP: fade due to nucleotidase inhibition and P2-receptor-mediated adenosine release.

When ATP or the related stable analogue, betagamma-imidoATP, were applied to rat hippocampal slices showing population spikes larger than 5 mV peak-to-peak amplitude, a depression of spike size was obtained, which showed a marked fade during the 10-min period of superfusion. The inhibitory responses were prevented by adenosine deaminase or 8-phenyltheophylline. Adenosine responses showed no fade. alphabeta-MethyleneADP enhanced the fade, while suramin at 50 micrometer prevented the early component of the responses. The results suggest that in slices with large population spikes, inhibitory responses to nucleotides are partly due to their conversion to adenosine, and partly due to the activation of P2 receptors which trigger the release of endogenous adenosine.

Suramin-sensitive suppression of paired-pulse inhibition by adenine nucleotides in rat hippocampal slices.

In order to assess the possible presence of presynaptic P2 receptors for nucleotides in the hippocampus, adenosine triphosphate and betagamma-methyleneATP have been examined on paired-pulse inhibition in rat hippocampal slices. Both compounds reproduced the effects of adenosine and reduced the amount of paired-pulse inhibition at an interpulse interval of 10 ms and increased the amount of facilitation at intervals of 20 and 50 ms. These effects were prevented by 8-phenyltheophylline and adenosine deaminase, indicating their mediation by adenosine. The effects were also reduced by suramin at 50 microM, suggesting the possible activation of P2 receptors. It is suggested that a population of P2 receptors may exist which promote the release of endogenous adenosine in the hippocampus.

Occlusive responses to adenosine A1 receptor and muscarinic M2 receptor activation on hippocampal presynaptic terminals.

There is substantial evidence for an interaction between adenosine A1 and muscarinic M1/M3 receptors in some tissues, either at the level of the receptors themselves or at the associated transduction system. We have now addressed the question of whether there is a similar interaction between A1 and presynaptic M2 receptors in the hippocampus. The effects of cyclopentyladenosine (CPA) were studied alone or in combination with the M2 receptor agonist oxotremorine-M. The ability of both to depress synaptic transmission presynaptically at the concentrations used was confirmed using paired-pulse inhibition. When combined at a range of concentrations, the effects of the two agents were less than additive, suggesting that they are acting by a common transduction system. The results indicate that the modulatory, antagonistic effects of A1 adenosine receptors are exerted not only on postjunctional M1/M3 receptors but also at M2 presynaptic receptors.