The effect of pulsed radiofrequency application on nerve healing after sciatic nerve anastomosis in rats.

The Effect of Pulsed Radiofrequency Application on Nerve Healing After Sciatic Nerve Anastomosis in Rats. In this study, we aimed to evaluate the histomorphological and functional effect of Pulsed Radiofrequency (PRF) application on regeneration after experimental nerve damage in rats. Forty Sprague-Dawley male rats were used in the study. Sciatic nerve incision was applied to all rats and then anastomosis was performed. Twenty rats were separated as the control group, and the remaining 20 rats underwent PRF every day at 42oC, for 120 seconds. The groups were divided into two further subgroups to be sacrificed on the 15th and 30th days. Tissue samples were obtained from all groups at 24 hours and 72 hours after the injury. Sections of sciatic nerve samples were stained with hematoxylin-eosin for light microscopic investigation and prepared for evaluation of ultrastructural changes with transmission electron microscopy. In the evaluation of axon numbers and diameters were seen that the 30th-day RF group had an increase compared to the control group. In the electron microscopic examination, it was observed that myelinated and unmyelinated nerve fiber sheaths had borders that are more regular in the RF group, the nucleus structures of schwann cells were better preserved, mitochondrial damage was less, and the extensions of fibroblast and collagen fibers were smoother than the control group. The findings suggested that PRF application has a positive contribution histologically on nerve healing in the early period after full-layer incision nerve injury anastomosis surgery.

Modulating the thermostability of Endoglucanase I from Trichoderma reesei using computational approaches.

In the last decades, effective cellulose degradation became a major point of interest due to the properties of cellulose as a renewable energy source and the widespread application of cellulases (the cellulose degrading enzymes) in many industrial processes. Effective bioconversion of lignocellulosic biomass into soluble sugars for ethanol production requires use of thermostable and highly active cellulases. The library of current cellulases includes enzymes that can work at acidic and neutral pH in a wide temperature range. However, only few cellulases are reported to be thermostable. In order to alleviate this, we have performed a hybrid approach for the thermostabilization of a key cellulase, Endoglucanase I (EGI) from Trichoderma reesei. We combined in silico and in vitro experiments to modulate the thermostability of EGI. Four different predictive algorithms were used to set up a library of mutations. Three thermostabilizer mutations (Q126F, K272F, Q274V) were selected and molecular dynamics simulations at room temperature and high temperatures were performed to analyze the effect of the mutations on enzyme structure and stability. The mutations were then introduced into the endoglucanase 1 gene, using site-directed mutagenesis, and the effect of the mutations on enzyme structure and stability were determined. MD simulations supported the fact that Q126F, K272F and Q274V mutations have a thermostabilizing effect on the protein structure. Experimental studies validated that all of the mutants exhibited higher thermostability compared with native EGI albeit with a decrease in specific activity.

Assesment of the reactions of caregivers of a cancer patients: validity and reliability of the scale of "Your reactions helping your family member" in Turkish society.

BACKGROUND: This study has been descriptively designed in order to evaluate the validity and reliability of the scale of "Your Reactions to Helping Your Family Member" for Turkish society. The scale that was developed by Archbold and Steward in 1983 being applied at caregivers is included within Family Care Inventory. MATERIALS AND METHODS: The scale which is adapted for Turkey was applied to 132 caregivers of cancer patients, who are family members, twice by face-to-face interview method. Sample of the study consists of 132 caregivers of cancer patients who had outpatient treatment in chemotherapy unit of a university hospital in Izmir. Quantitative and percentage Ugur and Fadiloglu tests were used in the analysis of descriptive data regarding caregivers, Kendall W analysis was used to assess content validity of the scale, Pearson's Product Moment Correlation was used for test-retest reliability of the scale and Cronbach Alpha Analysis was used for internal consistency. RESULTS: Validity of the scale was determined by using Kendall's Coefficient of Concordance (W: 0.62, p > 0.05) and it was evaluated as there's no difference ofopinion among the experts. Test-retest reliability coefficient between the first and the second application of the scale was found - as. 88. CONCLUSIONS: The scale which is ascertained to be valid and reliable as a result of our study can be used to assess the reactions of caregivers of cancer patients during the care giving period in Turkish society.

Modifying the catalytic preference of tributyrin in Bacillus thermocatenulatus lipase through in-silico modeling of enzyme-substrate complex.

In this study, rational design for Bacillus thermocatenulatus lipase (BTL2) was carried out to lower the activation barrier for hydrolysis of short-chain substrates. In this design, we used computational models for the enzyme-substrate (ES) complexes of tributyrin (C4) and tricaprylin (C8), which were generated through docking and molecular dynamics (MD) simulations. These ES complexes were employed in steered MD (SMD) simulations with Jarzynski's equality to estimate their relative binding free energies. Potential mutation sites for modifying the chain-length selectivity of BTL2 were found by inspecting the SMD trajectories and fine-tuning the volume and hydrophobicity of the cleft. Seven mutations (F17A, L57F, V175A, V175F, I320A, I320F and L360F) were performed to cover three binding pockets for sn-1, sn-2 and sn-3 acyl chains. The relative binding free energies of the mutant ES complexes formed by C4 and C8 ligands were calculated similarly. The experimental routines of protein engineering including site-directed mutagenesis, heterologous protein expression and purification were performed for all lipases. Steady-state specific activities towards C4 and C8 were determined for wild-type and mutant lipases, which gave an estimate of the relative change in the binding free energy of transition state complex (ES(‡)). The chain-length selectivity of mutants was determined from the relative changes in the activation barrier of hydrolysis of C4 and C8 triacylglycerol with respect to wild-type using computational and experimental findings. The most promising mutant for C4 over C8 preference was found to be L360F. We suggest that L360F may be at a critical position to lower the activation barrier for C4 and elevate it for C8 hydrolysis.

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis.

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.

From in silico to in vitro: modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris.

In this study, a major cellulase, namely endoglucanase 1 (EGI) from Trichoderma reesei was mutated by the introduction of four different lysine and glycine rich loops to create a hotspot for directed crosslinking of EGI away from the active site. The impact of the inserted loops on the stability of the enzyme was analyzed using molecular dynamics (MD) and the effect on the active site was studied using molecular mechanics (MM) simulations. The best loop mutation predicted in silico (EGI_L5) was introduced to EGI via site directed mutagenesis. The loop mutant EGI_L5 and EGI were both expressed in Pichia pastoris. Enzymes were characterized and their activities against soluble substrates such as CMC and 4-MUC were determined. Both enzymes exhibited similar pH and temperature activity and thermal stability profiles. Moreover, specific activity of EGI_L5 against 4-MUC was found to be the same as the native enzyme.

Clustering of protein families into functional subtypes using Relative Complexity Measure with reduced amino acid alphabets.

BACKGROUND: Phylogenetic analysis can be used to divide a protein family into subfamilies in the absence of experimental information. Most phylogenetic analysis methods utilize multiple alignment of sequences and are based on an evolutionary model. However, multiple alignment is not an automated procedure and requires human intervention to maintain alignment integrity and to produce phylogenies consistent with the functional splits in underlying sequences. To address this problem, we propose to use the alignment-free Relative Complexity Measure (RCM) combined with reduced amino acid alphabets to cluster protein families into functional subtypes purely on sequence criteria. Comparison with an alignment-based approach was also carried out to test the quality of the clustering. RESULTS: We demonstrate the robustness of RCM with reduced alphabets in clustering of protein sequences into families in a simulated dataset and seven well-characterized protein datasets. On protein datasets, crotonases, mandelate racemases, nucleotidyl cyclases and glycoside hydrolase family 2 were clustered into subfamilies with 100% accuracy whereas acyl transferase domains, haloacid dehalogenases, and vicinal oxygen chelates could be assigned to subfamilies with 97.2%, 96.9% and 92.2% accuracies, respectively. CONCLUSIONS: The overall combination of methods in this paper is useful for clustering protein families into subtypes based on solely protein sequence information. The method is also flexible and computationally fast because it does not require multiple alignment of sequences.

Left main coronary artery aneurysm in young patient with acute myocardial infarction.

Aneurysms of the left main coronary artery (LMCAA) are extremely uncommon, with an incidence of 0.1%. The main etiologic factor is atherosclerosis. Other causes include connective tissue disorders, trauma, vasculitis, congenital, mycotic, and idiopathic. These dilated sections of coronary artery are not benign pathology because they are subject to spasm, thrombosis, and subsequent distal embolism, spontaneous dissection and rupture. Treatment options include anticoagulation, custom-made covered stents, reconstruction, resection and exclusion with bypass. Our report on a young case illustrates the potential complication of LMCAA and presents its management.

Coupling of beta2-adrenoceptors to XLalphas and Galphas: a new insight into ligand-induced G protein activation.

Galpha(s) and extra-large Galpha(s) (XLalpha(s)) can both transduce receptor activation into intracellular cAMP generation. It is unknown, however, whether these two GNAS-locus products display distinct properties with respect to receptor coupling. Here, we show that XLalpha(s) couples to the beta2-adrenoceptor more efficiently than Galpha(s). In transfected human embryonic kidney 293 cells and mouse embryonic fibroblasts null for both Galpha(s) and XLalpha(s) (2B2 cells), basal cAMP accumulation mediated by XLalpha(s) was higher than that mediated by Galpha(s). Inverse agonist treatment reduced Galpha(s)-mediated basal activity, whereas its effect was markedly blunted on XLalpha(s)-mediated basal activity. Rank order of ligand efficacies regarding cAMP accumulation was the same when the receptor was coupled to XLalpha(s) or Galpha(s). However, ligand-induced and XLalpha(s)-mediated cAMP generation was higher than that mediated by Galpha(s). The relatively high efficiency of XLalpha(s)-mediated cAMP generation was conditional, disappearing with increased level of receptor expression or increased efficacy of ligand. Full-agonist responses in XLalpha(s)- and Galpha(s)-expressing cells were comparable even at low receptor levels, whereas partial agonist responses became comparable only when the receptor expression was increased (>3 pmol/mg). Radioligand binding studies showed that the high-affinity component in agonist binding to beta2-adrenoceptor was more pronounced in cells expressing XLalpha(s) than those expressing Galpha(s). We discuss these findings in the framework of current receptor-G protein activation models and offer an extended ternary complex model that can fully explain our observations.

Helicobacter pylori seropositivity in patients with acute coronary syndromes.

The aim of this study was to investigate the seroprevalence of Helicobacter pylori (HP) in patients with coronary atherosclerosis and acute coronary syndromes. We enrolled 152 patients (group I, 73 patients with acute coronary syndrome; group II, 79 patients with chronic stable angina) and 22 control subjects. An enzyme-linked immunosorbent assay for immunoglobulin (Ig) G test for HP diagnosis was used on all enrolled subjects. C-reactive protein (CRP) was also measured in all patients as an inflammatory marker. Seropositivity rates for HP were significantly higher in patients with coronary artery disease than in controls (80.2% versus 54.5%; P < 0.05). Although CRP level was significantly higher in group I than in group II (1.72 +/- 2.89 mg/dl versus 0.53 +/- 0.63 mg/dl, P < 0.0001), HP infection rates were similar between groups (86.3% versus 74.6%, P > 0.05). Infection with HP may influence atherogenesis through low-grade, persistent inflammatory stimulation. However, our data show evidence of increased systemic inflammation in patients with acute coronary syndrome, which was unrelated to an increased HP seropositivity.

The additional diagnostic value of a single-session combined scintigraphic and ultrasonographic examination in patients with thyroid and parathyroid diseases.

AIM: The aim of this study was to investigate the diagnostic efficacy and the clinical impact of scintigraphy combined with ultrasonography (USG) in the management of thyroid and parathyroid disorders in a large series of patients. METHODS: A total of 387 consecutive patients referred to the Nuclear Medicine Department of Hacettepe University in the period from January to September 2007 for investigating a thyroid (N. 339 patients: 232 females and 107 males, mean age+/-SD=48.9+/-13.6 years) or a parathyroid disease (N. 48 patients: 34 females and 14 males, mean age+/-SD=47.4+/-9.6 years) were prospectively evaluated, systematically performing both scintigraphy and USG in a single-day session. All the examinations were independently reviewed by two nuclear medicine physicians; in cases of discrepancy (3%) a final diagnosis was reached by consensus. For thyroid pathologies, USG results were considered to provide additional diagnostic information over scintigraphy: 1) if more nodules were identified; 2) if an irregular hyperactive area at scintigraphy suspicious for the presence of a nodule was clearly characterized at USG; 3) if a nodule missed at scintigraphy because of small size (<1 cm) was well depicted at USG, thus allowing an USG-guided fine needle aspiration cytology (FNAC) to reach a final diagnosis. For parathyroid pathologies, USG was considered to provide additional diagnostic information over scintigraphy if a low intensity radiotracer retention from the parathyroid suspected of being a parathyroid enlargement was clearly depicted at USG. In thyroid diseases, scintigraphy was considered to provide additional diagnostic information over USG, if the functional status of a diffuse or uni- or multi-nodular goiter were clearly defined at scintigraphy. In parathyroid diseases, scintigraphy was considered to provide additional diagnostic information over USG, if the differential diagnosis between a lymph node or a muscle or a vessel depicted at USG was clearly defined as a parathyroid enlargement at scintigraphy. Lastly, the clinical impact of the single-day combined scintigraphic/USG protocol was evaluated. RESULTS: USG. In the thyroid diseases group, USG was particularly useful: 1) to detect additional nodules in glands with suppressed thyroid tissue; 2) to disclose small thyroid nodules (<1 cm) in which it was possible to perform a USG-FNAC. In the parathyroid diseases group, USG was particularly useful for the detection of parathyroid enlargements not visualized at scintigraphy because characterized by a rapid wash-out of the radiotracer and thus by a low radioactivity intensity in the delayed scintigraphic images. Scintigraphy. In the thyroid diseases group, scintigraphy was particularly useful: 1) to diagnose a diffuse hyperfunctioning thyroid gland, and to differentiate in multinodular goiters the hyper- from the hypo-functioning nodules. In the hyperparathyroid diseases group, scintigraphy was particular useful in making a differential diagnosis between a true parathyroid enlargement vs. a lymph node or a muscle or a vessel as depicted at USG, and in cases with deeply or ectopically-positioned parathyroid glands. Combined imaging approach. Combined interpretation provided additional benefit in 225 of 339 patients (64.4%). Overall, using the combined scintigraphic/USG single-day protocol, in the thyroid diseases group the therapeutic strategy (drug therapy vs radioiodine therapy vs surgery) was changed in 176/225 patients (78.2%, P<0.001 by chi(2) of Pearson), and in the parathyroid disease group the therapeutic strategy (medical therapy vs surgery) was changed in 18/48 patients (37.5%, P<0.01 by chi2 test of Pearson). CONCLUSION: In agreement with some previous published experiences, the combined single-day scintigraphic/USG protocol systematically adopted in a large series of consecutive patients with thyroid and parathyroid diseases, enrolled in a limited period of time, proved to significantly increase the global diagnostic accuracy and to change the therapeutic strategy in more than two third of patients with a thyroid disease and in more than one third of patients with a parathyroid disease.

Vascular endothelial functions, carotid intima-media thickness, and soluble CD40 ligand levels in dipper and nondipper essential hypertensive patients.

OBJECTIVE: The lack of nocturnal decline in blood pressure (BP) is associated with an increase in cardiovascular events. Soluble CD40 ligand (sCD40L) is involved in the pathogenesis of risk factor-related vascular damage. The purpose of this study was to examine the relationship between vascular endothelial functions, carotid intima-media thickness (cIMT), plasma sCD40L levels and circadian BP profile in patients with essential hypertension. MATERIAL AND METHODS: The study population consisted of 81 essential hypertensive out-patients. BP dipping was defined as a night-to-day systolic and diastolic decrease >or=10%. Forty-seven dipper and 34 nondipper patients were compared. High sensitivity C-reactive protein (hs-CRP), sCD40L and urinary albumin were measured. Brachial artery flow-mediated dilatation (FMD) and cIMT was compared between the groups. RESULTS: sCD40L level (3.28 +/- 2.08 and 2.30 +/- 1.99 ng/ml, respectively, P = 0.036) and urinary albumin concentration (36.7 +/- 20.1 and 23 +/- 29.7 mg/l, respectively, P < 0.0001) were higher in nondippers than in dippers. Serum hs-CRP levels were not significantly different. FMD was found higher in dippers than nondippers (11.8 +/- 3.9% and 6.6 +/- 2.2%, respectively, P < 0.0001). The average cIMT was significantly higher in nondippers than dippers (0.928 +/- 0.060 Vs. 0.734 +/- 0.134 mm; P < 0.0001). CONCLUSIONS: Nondipper patern has an additional negative effect on endothelial functions in hypertensive patients. Nondippers have enhanced sCD40L levels, which may contribute to their increased susceptibility to develop vascular damage.

Nuclear medicine techniques for radio-guided surgery of hyperparathyroidism.

In recent years there has been a prominent change in surgical trends from extensive approaches to minimally invasive techniques in hyperparathyroidism. In contrast to classical bilateral exploration methods, minimally invasive parathyroidectomy necessitates an adequate preoperative imaging modality, in order to focus exactly the lesion in interest. (99m)Tc-MIBI, as a highly sensitive and widely available radiopharmaceutical, provides high success rates in terms of preoperative parathyroid lesion localization. Besides preoperative imaging, minimally invasive parathyroidectomy also requires an intraoperative modality which can act as a guide to surgeon. Hand-held gamma probes are simply-operated lesion localization devices which serve as successful intraoperative guides to surgeon. (99m)Tc-MIBI based minimal invasive parathyroidectomy has replaced the traditional 4 gland exploration as the procedure of choice in many institutions with comparable cure rates.

Use of plasma Epstein-Barr virus DNA monitoring as a tumor marker in follow-up of patients with nasopharyngeal carcinoma: preliminary results and report of two cases.

Recent studies suggest that plasma Epstein-Barr virus (EBV) DNA may reflect tumor burden in patients with nasopharyngeal cancer. A prospective study was initiated to investigate this correlation in 125 patients (34 pretreatment [Group A], 78 in remission [Group B] and 13 relapsed [Group C]) and 19 healthy controls. In group A, EBV DNA was detected in plasma samples of 24 (70%) patients. In Group B, EBV DNA was detected in 7 patients (range 77-13,731 copies/mL) and further imaging in all but one of these patients revealed active disease confirmed by ultrasound-guided fine-needle biopsy. There was only one false-positive case; this patient is currently under follow-up. Here we describe 2 of the 7 patients with detectable plasma EBV DNA in whom recurrence was documented by PET scan during follow-up. Our results showed that in group B the positive predictive value of quantitative analysis of plasma EBV DNA was 85%. Quantitative analysis of EBV DNA in plasma seems to become an integral part of screening, staging, monitoring, and prediction of relapse in patients with nasopharyngeal carcinoma. However, previous studies cannot be considered definitive and more reports on the use of this technique are urgently needed from both endemic and non-endemic regions.

Percutaneous septal ablation for left mid-ventricular obstructive hypertrophic cardiomyopathy: a case report.

BACKGROUND: Mid-ventricular obstructive hypertrophic cardiomyopathy (MVOHC) is a rare type of cardiomyopathy. The diagnosis is based on the hourglass appearance on the left ventriculogram and the presence of pressure gradient between apical and basal chamber of the ventriculum on the hemodynamic assessment. CASE PRESENTATION: The present case represents successful percutaneous treatment with septal ablation to patient with MVOHC associated with systolic anterior motion of the mitral valve and obstruction at both the mid-ventricular and outflow levels. CONCLUSION: Alcohol septal ablation has been proposed as less invasive alternatives to surgery in patients with MVOHC.

Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling.

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.

Comparison of In-111 octreotide and Tc-99m (V) DMSA scintigraphy in the detection of medullary thyroid tumor foci in patients with elevated levels of tumor markers after surgery.

PURPOSE: In this retrospective study, the authors evaluated the utility of In-111 octreotide (OctreoScan) and Tc-99m (V) DMSA scintigraphy for the localization of recurrent metastatic tumor foci in patients with medullary thyroid cancer (MTC) and compared the findings with those of conventional radiologic imaging methods. METHODS: The scintigraphic images were compared with computed tomography (CT) and magnetic resonance imaging (MRI) and ultrasonography (US) in 14 patients (8 men, 6 women; age range, 22 to 74 years) with elevated calcitonin and carcinoembryonic antigen levels after total thyroidectomy. All scintigraphic image findings were evaluated qualitatively as mild uptake (+) and moderate to marked uptake (++). RESULTS: In-111 octreotide may be superior to Tc-99m (V) DMSA for the detection of tumor foci of patients with MTC on a patient basis (78.5% versus 57.1%) and on a lesion basis (44.1% versus 30.2%). The sensitivity rate for In-111 octreotide (78.5%) was also similar to that of CT and MRI on a patient basis. Conversely, the combined use of Tc-99m (V) DMSA and In-111 octreotide revealed the best sensitivity rate (85.7%) on a patient basis, whereas the combined use of CT and MRI showed the best sensitivity rate (81.3%) on a lesion basis. CONCLUSIONS: These findings suggest that In-111 octreotide is superior to Tc-99m (V) DMSA and has a similar sensitivity rate to CT and MRI for the diagnosis of recurrent or metastatic MTC. Although the combined use of In-111 octreotide and Tc-99m (V) DMSA was most sensitive, the combined use of CT and MRI with radionuclide imaging methods may better detect more metastatic tumor foci.