Evaluation of the melting temperature of TaqMan probes as a genotyping method for IDH1, IDH2, and H3F3A in pediatric astrocytomas / Evaluación del método de temperatura de disociación de sondas TaqMan para la genotipificación de IDH1, IDH2 y H3F3A en astrocitomas pediátricos

Abstract Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-TaqMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

Resumen Introducción: Los astrocitomas son un tipo de cáncer que afecta al sistema nervioso central y representan el tumor sólido más común durante la infancia. En el año 2016, la Organización Mundial de la Salud estableció un sistema de clasificación molecular para reagrupar tumores con identidades genéticas similares y lograr un diagnóstico más preciso, lo que lleva a tomar las decisiones clínicas idóneas al elegir el tratamiento de pacientes con este tipo de tumores. Métodos: Se evaluó un protocolo que involucra el uso de sondas TaqMan en un ensayo de reacción en cadena de la polimerasa asimétrica para la detección de mutaciones en astrocitomas. Se secuenciaron cuatro astrocitomas difusos (Grado II), tres astrocitomas anaplásicos (Grado III) y cuatro glioblastomas (Grado IV). Se intentó establecer las condiciones del análisis para la genotipificación de los astrocitomas pediátricos mediante la identificación de las temperaturas de disociación específicas de las sondas TaqMan producidas por la prescencia de las secuancias WT en los genes isocitrato deshidrogenasa 1 y 2 (IDH1, IDH2) y H3.3 histona A (H3F3A). Resultados: Los astrocitomas mostraron la secuencia wild type (WT) (silvestre) de los genes. Se utilizó una sonda TaqMan IDH1 para identificar el estado de este gen en dos templados WT de DNA (astrocitoma pilocítico y difuso) y se obtuvieron cuatro valores de temperatura de disociación (65.6-92.2 °C). Solo cuatro de las 29 reacciones mostraron amplificación de DNA. La secuenciación de Sanger fue más rápida y confiable para detectar el estado de los genes en todas las muestras. Conclusiones: La secuenciación de Sanger sigue siendo la técnica más práctica para la genotipificación de astrocitomas pediátricos.

Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations.</p><p><b>RESULTS</b>A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing.</p><p><b>CONCLUSION</b>The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.</p>

Post space preparation timing of root canals sealed with AH Plus sealer

OBJECTIVES: To determine the optimal timing for post space preparation of root canals sealed with epoxy resin-based AH Plus sealer in terms of its polymerization and influence on apical leakage. MATERIALS AND METHODS: The epoxy polymerization of AH Plus (Dentsply DeTrey) as a function of time after mixing (8, 24, and 72 hours, and 1 week) was evaluated using Fourier transform infrared (FTIR) spectroscopy and microhardness measurements. The change in the glass transition temperature (Tg ) of the material with time was also investigated using differential scanning calorimetry (DSC). Fifty extracted human single-rooted premolars were filled with gutta-percha and AH Plus, and randomly separated into five groups (n = 10) based on post space preparation timing (immediately after root canal obturation and 8, 24, and 72 hours, and 1 week after root canal obturation). The extent of apical leakage (mm) of the five groups was compared using a dye leakage test. Each dataset was statistically analyzed by one-way analysis of variance and Tukey's post hoc test (α = 0.05). RESULTS: Continuous epoxy polymerization of the material with time was observed. Although the T(g) values of the material gradually increased with time, the specimens presented no clear T(g) value at 1 week after mixing. When the post space was prepared 1 week after root canal obturation, the leakage was significantly higher than in the other groups (p < 0.05), among which there was no significant difference in leakage. CONCLUSIONS: Poor apical seal was detected when post space preparation was delayed until 1 week after root canal obturation.

Root surface temperatura variation during mechanical removal of root canal filling material: an in vitro study / Variaciones térmicas en la superficie radiculardurante la desobturación mecánica del conducto. estudio in vitro

The aim of this study was to analyze in vitro temperature changeson the outer surface of the dental root during mechanical fillingremoval procedures.Thirty recently extracted single­rooted lower premolars were cuttransversally at 16 mm from the apex in order to standardizesample length. Endodontic treatment was performed on them. Thefilling material was subsequently removed using Gates Glidden(G1, G2, G3); Peeso (P1, P2, P3) and PostecPlus FRC (FRC)reamers while temperatures were measured on the outer surfaceusing a digital device with thermocouple at 0, 2, 4, 6, 8, 10 and 15seconds. Temperatures were compared using repeated measuresANOVA followed by pairwise comparison with Tukey’s test.All reamers caused significant temperature variation betweendifferent times (p<0.05). Pairwise comparisons indicated thattemperature increased with time for all reamers (p<0.05).Significant differences in temperature were found betweendifferent reamers after 0, 2, 4, 6, 8, 10 and 15 seconds (p<0.05)...

El objetivo del presente trabajo fue estudiar los cambios térmicosin vitro en la superficie externa de la raíz del diente, generadosdurante los procedimientos de desobturación mecánica.Se utilizaron 30 premolares inferiores unirradiculares reciente ­mente extraídos, que fueron seccionados transversalmente a16 mm del ápice para estandardizar la longitud de lasmuestras. Se realizó luego su tratamiento endodóntico. Lasmediciones de temperatura fueron realizadas mediante undispositivo digital con termocupla, en la superficie externa adiferentes tiempos (t = 0, 2, 4, 6, 8, 10 y 15 segundos), durantela desobturación con fresas de Gates Glidden (G1, G2, G3);de Peeso (P1, P2, P3) y la correspondiente a PostecPlus FRC(FRC). Los registros de temperatura fueron comparadosmediante pruebas de ANOVA de medidas repetidas, seguidaspor comparaciones entre pares mediante la prueba de Tukey.Con todas las fresas se encontró una variación significativa dela temperatura entre los diferentes tiempos (p<0.05). Las compa ­raciones entre pares indicaron que la temperatura se incrementócon el tiempo en todas las fresas (p<0.05). Se detectarondiferencias significativas de temperatura entre diferentes fresasdespués de 0, 2, 4, 6, 8, 10 y 15 segundos (p<0.05)...

Rapid screening of MT3243A>G mutation in mitochondrial diabetes with high resolution melting curve analysis and pyrosequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid, accurate, noninvasive and low cost method for screening MT3243A>G mutation in mitochondrial diabetes.</p><p><b>METHODS</b>Blood, saliva, and urine sediment samples were collected from 6 patients with confirmed mitochondrial diabetes and 50 healthy controls from Shanghai Children's Hospital and Shanghai Sixth People's Hospital. The heterozygosity levels of MT3243A>G mutation in above samples were detected with pyrosequencing, and the data were compared. MT3243A>G mutations were rapidly screened with high resolution melting curve analysis (HRM) in the urine sediment samples of 1070 diabetic patients from 4 communities in Shanghai. Furthermore, pyrosequencing was used to validate the suspected positive samples, and the heterozygosity levels were also quantified.</p><p><b>RESULTS</b>Comparative experiments found that heterozygosity of MT3243A>G mutation was 2 to 7 times higher in urine sediment than in saliva and blood samples from the 6 patients with confirmed mitochondrial diabetes. However, the heterozygosity was slightly higher in saliva than blood samples. MT3243A>G mutation was not detected in the 50 healthy controls. Two samples with suspected MT3243A>G mutation were identified in the 1070 urine sediment samples of diabetes patients with HRM screening, which were validated by pyrosequencing. The heterozygosity of MT3243A>G mutation were 33.32% and 14.67% in the urine sediment samples, respectively.</p><p><b>CONCLUSION</b>Urine sediment samples can be used for rapid screening of MT3243A>G mutation for its ease to collect, noninvasiveness and higher level of heterozygosity. HRM is suitable for rapid screening for mitochondrian mutations for its low cost, while such mutations could be detected with sensitivity and accuracy by pyrosequencing.</p>

Porous Crosslinked Polycaprolactone Hydroxyapatite Networks for Bone Tissue Engineering

In this study, porous scaffolds were produced by a thermal crosslinking of polycaprolactone diacrylate in the presence of hydroxyapatite (HA) and particulate leaching technique with sodium chloride as the water soluble porogen for bone tissue engineering applications. The prepared scaffolds were characterized using techniques such as Field Emission Scanning Electron Microscopy, Differential Scanning Calorimetry, and Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. Moreover, dynamic mechanical properties were investigated using Dynamic Mechanical Thermal Analysis. The obtained scaffolds present a porous structure with interconnected pores and porosity around 73%. It was found that the incorporation of HA particles to polycaprolactone (PCL) matrix resulted in an increased crystallinity. Moreover, both the storage modulus (E′) and glass transition temperature (T(g)) increased, while the loss factor (tan δ) decreased due to the hindrance of the HA particles to the mobility of polymer segments. Cytocompatability of the scaffolds was assessed by MTT assay and cell attachment studies. Osteoconductivity of the scaffolds was investigated with cells alkaline phosphatase extraction. The levels of alkaline phosphatase activity were found to be higher for PCL/HA network scaffold than for PCL network scaffold. In addition, cytocompatibility of the PCL/HA network scaffold indicated no toxicity, and cells were attached and spread to the scaffold walls.

ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger / 中国天然药物

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.

Concurrent Down-Regulation of PTEN and NKX3.1 Expression in Iranian Patients with Prostate Cancer

ABSTRACT NKX3.1 and PTEN genes are involved in the development and progression of prostate cancer (PCa). Here, in line with other studies that correlated the expression of these two genes, we aimed at evaluating the expression pattern of these genes in clinical PCa samples. Collectively, 81 tissue samples including 45 human PCa and 36 benign prostatic hyperplasia (BPH) specimens were included in the study. The tissue samples were subjected to RNA extraction and subsequently to cDNA synthesis according to the kit manufacturer's protocol. Quantitative Real-Time PCR assay was performed for each sample in triplicate reactions. REST and SPSS software were used to statistically analyze PTEN and NKX3.1 gene expression data. Expression level of both NKX3.1 and PTEN genes was down-regulated in PCa samples compared to BPH samples. The relative expression ratio of PTEN and NKX3.1 was decreased to 0.155 and 0.003, respectively (P=0.000). The results of Chi-Square analysis revealed a significant correlation between the expression of these genes in both BPH and cancer groups (P=0.004 and 0.001, respectively). According to previous studies and our data, we concluded that the association between the down-regulation of PTEN and NKX3.1 genes contributed to the prostate tumorigenesis. This might highlight the interaction between the proteins encoded by these genes. Furthermore, this finding might be exploited for the development of innovative diagnostic and therapeutic approaches in PCa.

Preliminary investigation on polymorphs of two traditional Chinese medicine reference substances / 中国中药杂志

Crystal structures of chemical drugs has been being investigated widely. But few attention has been paid to polymorphs-phenomena of active ingredients from Traditional Chinese Medicine(TCM). Taking anhydrous dehydroandrographolide and hydrousprim-O-glucosylcimifugin as example, differences between TCM reference substances (RSs) with different crystal structures were discussed by using microscopy, melting point determination, differential thermal analysis (DTA) and infrared (IR) methods. The results showed that different crystal structures could lead to change of melting points, thermal behaviors and IR spectrum. It's indicated that polymorphs may be considered if different physicochemical properties were obtained when applying TCM RS. Differences of chemical properties of active ingredients from TCM with different crystal structures need further investigation.

Molecular identification of hairy antler by analysis of high resolution melting / 中国中药杂志

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.

In Situ Polymerization and Characterization of Hydroxyapatite/polyurethane Implanted Material / 生物医学工程学杂志

In order to improve the interfacial bonding strength of hydroxyapatite/polyurethane implanted material and dispersion of hydroxyapatite in the polyurethane matrix, we in the present study synthesized nano-hydroxyapatite/polyurethane composites by in situ polymerization. We then characterized and analyzed the fracture morphology, thermal stability, glass transition temperature and mechanical properties. We seeded MG63 cells on composites to evaluate the cytocompatibility of the composites. In situ polymerization could improve the interfacial bonding strength, ameliorate dispersion of hydroxyapatite in the properties of the composites. After adding 20 wt% hydroxyapatite into the polyurethane, the thermal stability was improved and the glass transition temperatures were increased. The tensile strength and maximum elongation were 6.83 MPa and 861.17%, respectively. Compared with those of pure polyurethane the tensile strength and maximum elongation increased by 236.45% and 143.30%, respectively. The composites were helpful for cell adhesion and proliferation in cultivation.

Effects of kaixin jieyu decoction on behavior and glial fibrillary acidic protein expression in cerebral hippocampus of a rat vascular depression model / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects and anti-depression mechanisms of Kaixin Jieyu Decoction (, KJD).</p><p><b>METHODS</b>The rat vascular depression (VD) model was established by ligation of bilateral common carotid arteries (LBCCA) combined with chronic unpredictable mild stress (CUMS). Forty Wistar rats were randomly divided into sham, VD model, VD + high-dose KJD [15.4 g/(kg·d) of crude drug], VD + medium-dose KJD [7.7 g/(kg·d) of crude drug], and VD + fluoxetine [2.4 mg/(kg·d)] groups (n=8 in each group), and the treatments lasted for 21 days. Changes of behavior and hippocampus pathology were observed. The level of glial fibrillary acidic protein (GFAP) protein and mRNA in hippocampus was detected respectively by immunohistochemistry and real-time polymerase chain reaction.</p><p><b>RESULTS</b>Compared with the sham group, rats in model group showed a variety of behavioral obstacles, including a significant reduction in sucrose consumption percentage, horizontal and vertical activity scores in open-field tests (P<0.05 or P<0.01), pathological damage like neuronal degeneration, necrosis, and a significant decrease of GFAP protein and mRNA in hippocampus (P<0.01); compared with the model group, rats in the high-dose KJD group, medium-dose KJD group and fluoxetine group obtained notable higher behavioral scores, and pathological injury lessened in hippocampus with a increased expression of GFAP protein and mRNA P<0.05 or P<0.01); compared with the medium-dose KJD group and fluoxetine group, GFAP mRNA in high-dose KJD group expressed higer (P<0.05).</p><p><b>CONCLUSION</b>LBCCA combined with CUMS may cause depression-like behavioral changes resulting in the VD model of rats whose depression state can be ameliorated by KJD, and the mechanism of cerebral protection is related possibly with promoting expression of GFAP in hippocampus.</p>

HRM identification of Chinese medicinal materials Mutong / 中国中药杂志

High resolution melting (HRM), an important technology for genotyping and mutation scanning, has broad prospects in the authentification of traditional Chinese medicine. This paper selected universal trnH-psbA primers and used HRM to establish a new methods for identification of Akebia herbs. PCR was conduct at the annealing temperature of 58 degrees C and 35 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further analyzed. The results showed the Tm values of Caulis Akebiae was (81.84 ± 0.16), (85.28 ± 0.16) degrees C and Caulis Clematidis Armandii was (83.22 ± 0.19) degrees C and Caulis Aristolochiae manshuriensis was (81.67 ± 0.14) degrees C, (84.24 ± 0.10) degrees C with 5-125 mg - L-' DNA template, 0.4 μmol x L(-1) primer, 2.0 mmol x L(-1) Mg2+. This method can achieve the authentification of Akebia herbs and is simple, fast, high-throughput, visual.

Transition temperature range of thermally activated nickel-titanium archwires

Objectives: The shape memory resulting from the superelasticity and thermoelastic effect is the main characteristic of thermally activated NiTi archwires and is closely related to the transition temperature range (TTR). The aim of this study was to evaluate the TTR of thermally activated NiTi archwires commercially available. Material and Methods: Seven different brands of 0.019"x0.025" thermally activated nickel-titanium archwires were tested as received by differential scanning calorimetry (DSC) over the temperature range from -100°C to 150°C at 10°C/min. Results: All thermally activated NiTi archwires analyzed presented stage transformation during thermal scanning with final austenitic temperature (Af) ranging from 20.39°C to 45.42°C. Three brands of NiTi archwires presented Af close to the room temperature and, this way, do not present properties of shape memory and pseudoelasticity that are desirable in clinical applications. Conclusions: The thermally activated NiTi archwires present great variability in the TTR and the elastic parameters of each NiTi archwire should be provided by the manufacturers, to allow achievement of the best clinical performance possible. .

Efeito da temperatura das amostras de leite na concentração de cálcio solúvel e de beta-caseína: interferência no teste de estabilidade frente ao etanol / Effect of milk sample temperature on the concentration of soluble calcium and beta-casein: interference with the ethanol stability test

O objetivo deste trabalho foi avaliar a variação do perfil proteico e do cálcio solúvel na coagulação do leite pelo etanol nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Amostras de leite de 61 animais foram avaliadas quanto à estabilidade ao etanol nas concentrações de 66 a 92 por cento (v/v) nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Três amostras, após 24 horas de armazenamento a 4ºC, foram ultracentrifugadas em quadruplicata (40.000 x g) a 4ºC e a 20ºC, respectivamente, por 60 minutos. Em seguida, o sobrenadante foi retirado e submetido à análise do cálcio solúvel pela técnica via úmida (digestão nitroperclórica) e leitura em espectrofotômetro de absorção atômica. O perfil proteico foi analisado pela técnica de eletroforese capilar empregando kit específico para determinação proteica. Os resultados mostraram uma correlação positiva entre o aumento da temperatura das amostras e a estabilidade do leite frente às diferentes concentrações de etanol. A porcentagem de cálcio solúvel no sobrenadante após ultracentrifugação foi maior nas amostras tratadas a 4ºC (P<0,05). As amostras ultracentrifugadas na temperatura de 4ºC apresentaram quantidades superiores de β-caseína no sobrenadante em comparação com as amostras tratadas a 20ºC. O abaixamento da temperatura favoreceu a migração da β-caseína e do cálcio coloidal para a fase solúvel do leite, o que possivelmente favoreceu o aumento da instabilidade das amostras no teste do etanol. Os resultados sugerem que a temperatura ideal para a realização de teste de estabilidade do leite frente ao etanol deveria ser de 21ºC...

The aim of this study was to evaluate the variation in protein profile and soluble calcium in milk coagulation by ethanol at 4ºC, 10ºC, 15ºC and 20ºC. Milk samples from 61 dairy cows were evaluated for stability of ethanol concentrations from 66 to 92 percent (v/v) at temperatures of 4°C, 10°C, 15°C and 20°C. Three samples were ultracentrifuged (40,000 x g) after 24 hours of storage at 4°C and 20°C, respectively, for 60 minutes. Their supernatants were removed and subjected to analyses of soluble calcium through nitro-perchloric digestion and atomic absorption spectrophotometry. The protein profiles were determined by capillary electrophoresis using a specific kit for protein determination. The results showed a positive correlation between the increase in temperature of the samples and the stability of milk against various concentrations of ethanol. The percentage of soluble calcium in the supernatant after centrifugation was higher in samples treated at 4°C (P<0.05). The samples ultracentrifuged at 4°C showed higher amounts of β-casein in the supernatant compared with samples stored at 20°C. The lowering of the temperature favored the migration of β-casein and colloidal calcium to the soluble phase of milk, which may also have favored the instability of milk in the ethanol test. According to the results, the milk sample temperature for the ethanol stability test should be 21ºC...

Rapid quantification of total nitrogen and end-point determination of hide melting in manufacturing of donkey-hide gelatin / 中国中药杂志

Hide melting presents itself as one of the most critical processes in the production of donkey-hide gelatin. Here a NIR-based method was established for the rapid analysis of in-process hide melting solutions as well as for end-point determination of this process. Near infrared (NIR) spectra of hide melting solutions were collected in transflective mode. With the contents of total nitrogen determined by the Kjeldahl method as reference values, partial least squares regression (PLSR) was employed to build calibration models between NIR spectra and total nitrogen. Model parameters including wavelength range and PLS factors were optimized to achieve best model performance. Based on the contents of total nitrogen predicted by calibration model, end point of hide melting was determined. The constructed PLS model gave a high correlation coefficient (R2) of 0.991 3 and a root mean square error of prediction (RMSEP) of 0.807 g x L(-1). With the predicted total nitrogen and predefined limit, decisions concerning the proper times of melting were made. This research demonstrated that NIR transflectance spectroscopy could be used to expeditiously determine the contents of total nitrogen which was subsequently chosen as the indictor for determining the end-point of hide melting. The proposed procedure may help avoid unnecessary raw material or energy consumption.

Melting curve aaalysis for the screening of heptitis b virus genotypes a, d annd f in patients from a general hospital in southern Brazil / Analise da curva de melting para o screening dos genotipos A, D e F do virus da hepatite B em pacientes de um hospital geral do sul do Brasil.

Context Hepatitis B virus (HBV) can cause fulminant hepatitis, cirrhosis and hepatocellular carcinoma, and is one of the most common causes of acute and chronic liver failure. The genetic variants of HBV can be decisive for the evolution of these diseases as well as for the election of therapy. Objectives The aim of this study was to evaluate and standardize an in house methodology based on the analysis of the melting curve polymerase chain reaction (PCR) of real-time (qPCR) to screen for genotypes A, D and F of HBV in patients from a hospital in Rio Grande do Sul, Brazil. Methods We evaluated 104 patients presumably with HBV chronic infection. Viral DNA was extracted from plasma and viral genotypes and different mutations were determined using PCR-based protocols. Results A PCR-based methodology was standardized for the analysis of genotypes A, D and F of HBV. The technique was based in a nested PCR with the final step consisting of a multiplex real-time PCR, using the melting curve as a tool for the differentiation of fragments. A higher frequency of genotype D (44.4%), followed by genotype A (22.2%) and genotype F (3.7%) was observed. Conclusion The standardized assay, a nested PCR-multiplex qPCR using specific primers, provides a rapid and accurate method for the differentiation of HBV genotypes that are more frequent in Southern Brazil – A, D and F. This method can be applied in the clinical practice. .

Contexto O vírus da hepatite B pode causar hepatite fulminante, cirrose e carcinoma hepatocelular, sendo uma das causas mais frequentes de doença aguda e crônica do fígado. As variantes genéticas do VHB podem ser determinantes para a evolução da doenças assim como para a eleição da terapêutica. Objetivos O objetivo deste estudo foi padronizar e avaliar uma metodologia “in house”, através da utilização da curva de melting de reação em cadeia da polimerase (PCR) em tempo real (qPCR), como rastreamento para análise dos genótipos A, D e F do vírus da hepatite B em pacientes do Rio Grande do Sul. Métodos Foram avaliados 104 pacientes supostamente com infecção crônica pelo VHB. O DNA foi extraído com kit comercial, os genótipos e as mutações foram determinados utilizando diferentes protolocos baseados em PCR. Resultados Foi padronizada uma metodologia baseada em PCR para a análise dos genótipos A, D e F do VHB. A técnica consistiu de uma PCR Nested incluindo uma etapa final de PCR em tempo real Multiplex, utilizando a curva de melting como ferramenta para a definição dos fragmentos. Foi observada uma maior frequência do genótipo D (44,4%), seguido do genótipo A (22,2%) e do genótipo F (3,7%) na amostra analisada. Conclusão O ensaio padronizado fornece um método rápido e preciso para diferenciar genótipos do VHB mais frequentes no sul do Brasil – A, D e F – usando um PCR Nested Multiplex com primers específicos, o qual apresenta potencial aplicação na prática clínica. .

Caracterização de resinas para base de prótese e reembasamento: propriedades físico-químicas e mecânicas / Characterization of denture base and reline resins: physical, chemical and mechanical properties

Este estudo avaliou propriedades físico-químicas e mecânicas de resinas para base de prótese, uma fotoativada (Eclipse - E) à base de UDMA e outra termicamente ativada por micro-ondas (Nature Cryl MC - NC) à base de PMMA, e um reembasador quimicamente ativado (Tokuyama Rebase Fast II - TR II) à base de PEMA. Propriedades viscoelásticas (E', tan δ e Tg), contração linear de polimerização (CLP), sorção (Sor) e solubilidade (Sol) em água e em saliva artificial, grau de conversão (GC) e coeficiente de inchamento (CI) foram avaliados para a caracterização dos polímeros. A resistência à flexão dos materiais intactos (E, NC, TR II) e reembasados (E-E, E-TRII, NC-NC- NC-TR II) também foi avaliada. As propriedades viscoelásticas foram avaliadas em corpos-de-prova retangulares (64 x 10 x 3,3 mm), submetidos (n=2) à análise térmica dinâmico-mecânica (DMTA) em dois ciclos de varredura após confecção (controle) e armazenagem em saliva. O módulo de armazenamento (E') e o amortecimento mecânico (tan δ) a 37 ºC e a temperatura de transição vítrea (Tg) foram registrados durante o 1º ciclo da DMTA. A CLP (%) foi avaliada (n=10), em projetor de perfil (Nikon 6C), em corpos-de-prova circulares (50 x 0,5 mm). A Sor e a Sol (µg/mm3) foram mensuradas (n=5) seguindo as recomendações da ISO 1567. O GC (%) foi avaliado (n=5) por espectroscopia de infravermelho por transformadas de Fourier (32 leituras; resolução 4,0 cm-1 ). O coeficiente de inchamento foi realizado (n=5) de acordo com a teoria de Flory-Rhener. Os ensaios mecânicos de flexão (0,5 mm/min) foram realizados (n=10) em corpos-de-prova (64 x 10 x 3,3 mm) intactos ou reembasados e a resistência (MPa) calculada na tensão máxima (RFM) e no limite de proporcionalidade (RFLP). Os dados foram avaliados por ANOVA, seguida por Tukey HSD (α=0,05) ou Dunnett (α=0,05). As resinas E e NC apresentaram similar E', superior a TR II. O tan δ foi ordenado como TR II > NC > E. A Tg não foi alterada após armazenagem em saliva artificial. Os maiores valores de Tg nos grupos controle e armazenados foram apresentados, respectivamente, pelos materiais E e NC. Houve deslocamento dos picos de tan δ após o segundo ciclo de DMTA. Independente do meio de imersão, a Sor (µg/mm3 ) foi ordenada em NC > E > TR II. A armazenagem em saliva reduziu a Sol (µg/mm3 ) dos materiais. A resina E exibiu os menores valores de CLP, CI e o maior GC. NC exibiu a maior CLP e CI. TR II exibiu valores intermediários de CLP, CI e o menor GC. Os maiores valores de RFM e RFLP foram produzidos por E. A RFM e RFLP dos materiais reembasados foi ordenada em E-E > NC-NC > E-TRII> NC-TR II. A armazenagem em saliva não influenciou a resistência à flexão dos materiais intactos ou reembasados. No geral, a resina para base de prótese E apresentou propriedades físico-químicas e mecânicas superiores à NC e TR II

This study evaluated the physical-chemical and mechanical properties of denture base resins, one UDMA light-activated (Eclipse-E) and one PMMA heat-polymerized (Nature Cryl MC - NC). The PEMA autopolymerizing reliner (Tokuyama Rebase Fast II - TR II) was also evaluated. Viscoelastic properties (E', tan δ e Tg), linear shrinkage polymerization (LPS), sorption (Sor) and solubility (Sol) in water and in artificial saliva, degree of conversion (DC) and coefficient of swelling (CS) were employed for polymer characterization. The flexural strength of intact (E, NC, TR II) and relined samples (E-E, E-TRII, NC-NC- NC-TR II) was also made. The viscoelastic properties were evaluated by two cycles of dynamic mechanical thermal analysis (DMTA) on each rectangular (64 x 10 x 3.3 mm) samples (n=2). The storage modulus (E') and loss tangent (tan δ) at 37 ºC and the glass transition temperature (Tg) were recorded from the 1st DMTA cycle. LPS (%) was evaluated (n=10) by a Nikon 6C optical comparator, on circular samples (50 x 0.5 mm). Sor and Sol (µg/mm3 ) were measured (n=6) according to ISO 1567. DC (%) was evaluated (n=5) by FTIR spectroscopy (32 scans; resolution 4.0 cm-1). CS was evaluated (n=5) by Flory-Rhener theory. A three-point flexural tests (0.5 mm/min) was applied (n=10) on rectangular intact and relined samples. The ultimate flexural strength (FSU) and the flexural strength at the proportional limit (FSPl) were recorded in MPa. Data were analyzed with ANOVA, followed by Tukey HSD (α=0.05) ou Dunnett (α=0.05). E and NC had similar E', higher than TR II. Tan δ was arranged as TR II > NC > E. The Tg was not affected after saliva storage. The higher Tg values were obtained for E and NC. Tan δ peaks were shifted after 2 DMTA cycles. Regardless the medium immersion, Sor (µg/mm3 was arranged as NC > E > TR II. Saliva storage reduced Sol (µg/mm3) of the materials. E showed the lowest values of LPS, CS and the highest value of DC. NC showed the highest LPS and CS.TR II showed intermediated values of LPS, CS and the lowest DC. The highest FSU and FSPl values were exhibited by E. The FSU and FSPl of relined samples were arranged as E-E > NC-NC > E-TRII > NC-TR II. Saliva storage did not affect the flexural strength of intact and relined samples. Overall, the denture base resin E showed higher physicochemical and mechanical properties than NC and TR II

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Rapid screening for MTHFR gene 677C>T polymorphism in Down syndrome using high resolution melting curve and pyrosequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing.</p><p><b>METHODS</b>Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM.</p><p><b>RESULTS</b>Heterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%).</p><p><b>CONCLUSION</b>Respectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.</p>