Serum microRNA array analysis identifies miR-140-3p, miR-33b-3p and miR-671-3p as potential osteoarthritis biomarkers involved in metabolic processes.

BACKGROUND: MicroRNAs (miRNAs) in circulation have emerged as promising biomarkers. In this study, we aimed to identify a circulating miRNA signature for osteoarthritis (OA) patients and in combination with bioinformatics analysis to evaluate the utility of selected differentially expressed miRNAs in the serum as potential OA biomarkers. METHODS: Serum samples were collected from 12 primary OA patients, and 12 healthy individuals were screened using the Agilent Human miRNA Microarray platform interrogating 2549 miRNAs. Receiver Operating Characteristic (ROC) curves were constructed to evaluate the diagnostic performance of the deregulated miRNAs. Expression levels of selected miRNAs were validated by quantitative real-time PCR (qRT-PCR) in all serum and in articular cartilage samples from OA patients (n = 12) and healthy individuals (n = 7). Bioinformatics analysis was used to investigate the involved pathways and target genes for the above miRNAs. RESULTS: We identified 279 differentially expressed miRNAs in the serum of OA patients compared to controls. Two hundred and five miRNAs (73.5%) were upregulated and 74 (26.5%) downregulated. ROC analysis revealed that 77 miRNAs had area under the curve (AUC) > 0.8 and p < 0.05. Bioinformatics analysis in the 77 miRNAs revealed that their target genes were involved in multiple signaling pathways associated with OA, among which FoxO, mTOR, Wnt, pI3K/akt, TGF-ß signaling pathways, ECM-receptor interaction, and fatty acid biosynthesis. qRT-PCR validation in seven selected out of the 77 miRNAs revealed 3 significantly downregulated miRNAs (hsa-miR-33b-3p, hsa-miR-671-3p, and hsa-miR-140-3p) in the serum of OA patients, which were in silico predicted to be enriched in pathways involved in metabolic processes. Target-gene analysis of hsa-miR-140-3p, hsa-miR-33b-3p, and hsa-miR-671-3p revealed that InsR and IGFR1 were common targets of all three miRNAs, highlighting their involvement in regulation of metabolic processes that contribute to OA pathology. Hsa-miR-140-3p and hsa-miR-671-3p expression levels were consistently downregulated in articular cartilage of OA patients compared to healthy individuals. CONCLUSIONS: A serum miRNA signature was established for the first time using high density resolution miR-arrays in OA patients. We identified a three-miRNA signature, hsa-miR-140-3p, hsa-miR-671-3p, and hsa-miR-33b-3p, in the serum of OA patients, predicted to regulate metabolic processes, which could serve as a potential biomarker for the evaluation of OA risk and progression.

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

BACKGROUND: Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. METHODS: A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. RESULTS: In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. CONCLUSIONS: The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples.

Second line chemotherapy with methotrexate and gemcitabine in patients with relapsing head and neck cancer.

PURPOSE: The aim of this study was to evaluate the efficacy and toxicity profile of the combination of methotrexate and gemcitabine given as second-line treatment in patients with relapsing and/or metastatic head and neck cancer (HNC). PATIENTS AND METHODS: A total of 21 patients with HNC who had relapsed after first-line treatment with cisplatin-containing regimens were enrolles. Treatment consisted of intravenous (i.v.) administration of methotrexate 30 mg/m(2) and gemcitabine 800 mg/m(2) on days 1, 8, and 15 in cycles of 28 days. Primary sites included the larynx, tongue, nasopharynx, hypopharynx, nasal cavity, and parotid gland. The study end point was the evaluation of treatment efficacy and toxicity. RESULTS: Seven (33%) patients received only 1 or 2 cycles and discontinued treatment because of disease progression. Among 14 patients evaluable for respone, 1 complete (CR) and 2 partial responses (PR) were observed, yielding a response rate of 21.4%. The patient with CR remains relapse-free for 74(+) months. The 2 PR patients relapsed after 14 and 25 months and are still alive at 18 and 32 months, respectively. Seven patients showed minor response (MR) or stable disease (SD) with symptomatic relief lasting 4-12 months (mean 8). All but 2 of adequately treated patients (12/14 or 85.7%) attained a clinical benefit response (CBR). Mean time to progression (TTP) of all patients was 8 months (range 1-74(+)), while mean overall survival (OS) was 14 months (range 1-74(+)). Toxicity was mild to moderate and easily manageable. CONCLUSION: Methotrexate and gemcitabine combination is an effective second-line treatment for patients with relapsing and/or metastatic HNC. Moreover, this regimen is well tolerated with mild to moderate, easy to treat, toxicity.

Determination of ethylenediamine tetraacetic acid in injection forms by ion-pair chromatography.

A high performance liquid chromatographic method was developed for the determination of ethylenediamine tetraacetic acid (EDTA) in injection forms. The method consists of direct extraction of the samples with ethyl acetate; the organic layers were evaporated to dryness and further diluted to a 0.025% (w/v) copper nitrate in order to achive the formation of the EDTA-copper solution complex. The chromatographic separation was performed on a C8 Hypersil column. The mobile phase consisted of a mixture of acetonitrile-0.015 M tetrabutylammonium hydroxide (10:90, v/v), (pH* 7.0) pumped at a flow rate of 1.5 ml min(-1). The UV detector was operated at 300 nm. Correlation coefficients of the calibration graphs were better than 0.9995, relative standard deviation was less than 2.5%. Detection limit of EDTA was found to be 1.97 microg ml(-1).

Kinetics of the acidic and enzymatic hydrolysis of benazepril HCl studied by LC.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed and validated for the kinetic investigation of the chemical and enzymatic hydrolysis of benazepril hydrochloride. Kinetic studies on the acidic hydrolysis of benazepril hydrochloride were carried out in 0.1 M hydrochloric acid solution at 50, 53, 58 and 63 degrees C. Benazepril hydrochloride appeared stable in a pH 7.4 phosphate buffered solution at 37 degrees C and showed susceptibility to undergoing in vitro enzymatic hydrolysis with porcine liver esterase (PLE) in a pH 7.4 buffered solution at 37 degrees C. Benazeprilat appeared to be the major degradation product in both (chemical and enzymatic) studies of hydrolysis. Statistical evaluation of the proposed HPLC methods revealed their good linearity and reproducibility. Relative standard deviation (R.S.D.) was less than 4.76, while detection limits for benazepril hydrochloride and benazeprilat were 13.0 x 10(-7) and 9.0 x 10(-7) M, respectively. Treatment of the kinetic data of the acidic hydrolysis was carried out by non-linear regression analysis and k values were determined. The kinetic parameters of the enzymatic hydrolysis were determined by non-linear regression analysis of the data using the equation of Michaelis-Menten.

Teachers' causal attributions, cognitive, emotional and behavioural responses to students with emotional and behavioural difficulties.

BACKGROUND: The vast majority of children with emotional and behavioural difficulties are educated in their usual classes in ordinary schools across the countries. The study, therefore, of teachers' responses to these children is important for both teachers' and students' well-being in schools. Research provides evidence that teachers attribute students' difficulties to factors external to themselves, that is family or child factors and although they experience feelings of irritation and indifference with disruptive students they feel responsible and committed to help them. As far as their coping strategies are concerned, teachers seem to mostly favour positive interventions. AIMS: This study aimed to examine Greek teachers' causal attributions, emotional and cognitive responses, coping strategies and suggestions for effective coping strategies with students with emotional and behavioural difficulties. SAMPLE: This comprised 391 elementary teachers teaching in 60 public schools in the area of Athens. METHOD: Teachers completed an inventory presenting six short scenarios of students with emotional and behavioural difficulties, varying in the type of difficulty (conduct or emotional) and the degree of severity for teachers to handle them (mild or severe). RESULTS: Repeated-measures ANOVA revealed that teachers perceived school and teacher factors as causal of emotional and behavioural difficulties. Teachers expressed feelings of sympathy for these children, and perceived themselves as responsible, self-efficacious and inclined to help them. Finally, they reported using supportive techniques to handle emotional and behavioural difficulties, which they also considered as effective. CONCLUSIONS: Teacher training becomes crucial as a process of adopting patterns of thought and strategies for responding to students with emotional and behavioural difficulties.

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography.

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO4), allows a quantification limit of 2.52 microg ml(-1) and a detection limit of 0.83 microg ml(-1). Recoveries and absolute recoveries of cefepime from plasma were 96.13-99.44% and 94-102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 microg ml(-1) (n=10). The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.

Simultaneous determination of benazepril hydrochloride and hydrochlorothiazide by micro-bore liquid chromatography.

A micro-bore liquid chromatographic method was developed for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in pharmaceutical dosage forms. The use of a BDS C-18 micro-bore analytical column, results in substantial reduction in solvent consumption and increased sensitivity. The mobile phase consisted of a mixture of 0.025 M sodium dihydrogen phosphate (pH 4.8) and acetonitrile (55:45, v/v), pumped at a flow rate of 0.40 ml min(-1). Detection was set at 250 nm using an ultraviolet detector. Calibration graphs are linear (r better than 0.9991, n = 5), in concentration range 5.0-20.0 microg ml(-1) for benazepril hydrochloride and 6.2-25.0 microg ml(-1) for hydrochlorothiazide. The intra- and interday R.S.D. values were <1.25% (n = 5), while the relative percentage error (Er) was <0.9% (n = 5). The detection limits attained according to IUPAC definition were 0.88 and 0.58 microg ml(-1) for benazepril hydrochloride and hydrochlorothiazide, respectively. The method was applied in the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Kinetic study on the degradation of prazepam in acidic aqueous solutions by high-performance liquid chromatography and fourth-order derivative ultraviolet spectrophotometry.

A reversed-phase HPLC method was developed for the kinetic investigation of the acidic hydrolysis of prazepam which was carried out in hydrochloric acid solutions of 0.01, 0.1 and 1.0 M. In addition, a fourth-order derivative method for monitoring the parent compound itself was proposed and evaluated. One intermediate was observed by HPLC, which should be formed from breakage of the azomethine linkage. Further slow hydrolysis of the amide bond led to the benzophenone product that was isolated and identified. The mechanism of hydrolysis was biphasic, showing a consecutive reaction with a reversible step. Relative standard deviation was less than 2% for HPLC and less than 5% for the derivative method. Detection limits were 1.2 x 10(-7) M for the former method and 6.7 x 10(-7)M for the latter. Accelerated studies at higher temperatures were employed. Results of HPLC and fourth-order derivative methods were statistically the same.

Determination of piroxicam and its major metabolite 5-hydroxypiroxicam in human plasma by zero-crossing first-derivative spectrophotometry.

A zero-crossing first-derivative spectrophotometric method for the determination of piroxicam and its major metabolite 5-hydroxypiroxicam (5-HP) in human plasma is described. This technique permits the quantification of compounds with closely overlapping spectral bands without any separation step. The method consists of direct extraction of the less-ionised forms of piroxicam and 5-hydroxypiroxicam with pure diethyl ether. First derivative values at 343.5 and 332.5 nm for piroxicam and 5-HP, respectively, were obtained. The absolute recovery of the method was found to be 89.4% for piroxicam and 90.3% for 5-HP. Calibration graphs are linear (r better than 0.9998), with zero-intercept, in the concentration range 0.5-12.0 micrograms ml-1 for both compounds. The limits of quantification attained according to the IUPAC definition were 0.29 and 0.27 micrograms ml-1 for piroxicam and 5-HP, respectively. The results obtained by the proposed method were in good agreement with those found by the high-performance liquid chromatographic method (HPLC).

Acidic hydrolysis of bromazepam studied by high performance liquid chromatography. Isolation and identification of its degradation products.

A kinetic study on the acidic hydrolysis of bromazepam was carried out in 0.01 M hydrochloric acid solution at 25 and 95 degrees C. A reversed-phase HPLC method was developed and validated for the determination of bromazepam and its degradation products. Bromazepam degraded by a consecutive reaction with a reversible first step. Two degradation products were isolated and identified by infrared, 1H and 13C nuclear magnetic resonance and mass spectroscopy. Spectroscopic data indicated that N-(4-bromo-2-(2-pyridylcarbonyl)phenyl)-2-aminoacetamide was the intermediate degradation product of this acid hydrolysis, whereas 2-amino-5-bromophenyl-2-pyridylmethanone was the final one. Therefore, the mechanism of this acid-catalysed hydrolysis involved initial cleavage of the 4,5-azomethine bond, followed by slow breakage of the 1,2-amide bond. Statistical evaluation of the HPLC method revealed its good linearity and reproducibility. Detection limits were 3.8 x 10(-7) M for bromazepam, 6.25 x 10(-7) M for the intermediate and 8.16 x 10(-7) M for the benzophenone derivative.

Second-derivative spectrophotometric determination of naproxen in the presence of its metabolite in human plasma.

A second-derivative spectrophotometric method for the determination of naproxen in the absence or presence of its 6-desmethyl metabolite in human plasma is described. The method consists of direct extraction of the non-ionized form of the drug with pure diethyl ether and determination of the naproxen by measuring the peak amplitude (mm) in the second-order derivative spectrum at a wavelength of 328.2 nm. The efficiency of the extraction procedure expressed by the absolute recovery was 94.6 +/- 0.7% (mean +/- s) for the concentration range tested, and the limit of quantification attained according to the IUPAC definition was 2.42 mg l-1. The linear dynamic range for naproxen was 5.0-100.0 mg l-1, the correlation coefficient for the calibration graphs was excellent, r = 0.99993 (n = 6), the precision (Sr) was better than 4.58% and the accuracy was satisfactory (Er < 2.32%). The results obtained by the proposed method were in good agreement with those found by an HPLC method.

Simultaneous determination of clopamide-pindolol combination in tablets by zero-crossing derivative spectrophotometry.

A first-derivative spectrophotometric method, using a 'zero-crossing' technique of measurement has been used for determining clopamide-pindolol mixture in tablets. In the first-derivative mode the zero-crossing points of clopamide and pindolol occur at 272.6 and 262.4 nm, respectively. The relative ease offered by this technique for the quantification of these drugs with closely overlapping bands was demonstrated. The linearity of the calibration curves was satisfactory (r = 0.9998) and the precision (RSD%) better than 1.89. Detection limits were 0.50 and 0.44 micrograms ml-1 for pindolol and clopamide, respectively. No spectral interferences from tablet excipients were found. Applications are given for the assay of commercial tablets and content uniformity test. The procedures proved to be suitable for rapid and reliable quality control.