High-resolution manometry and impedance-pH/manometry: valuable tools in clinical and investigational esophagology

Both high-resolution manometry (HRM) and impedance-pH/manometry monitoring have established themselves as research tools and both are now emerging in the clinical arena. Solid-state HRM capable of simultaneously monitoring the entire pressure profile from the pharynx to the stomach along with pressure topography plotting represents an evolution in esophageal manometry. Two strengths of HRM with pressure topography plots compared with conventional manometric recordings are (1) accurately delineating and tracking the movement of functionally defined contractile elements of the esophagus and its sphincters, and (2) easily distinguishing between luminal pressurization attributable to spastic contractions and that resultant from a trapped bolus in a dysfunctional esophagus. Making these distinctions objectifies the identification of achalasia, distal esophageal spasm, functional obstruction, and subtypes thereof. Ambulatory intraluminal impedance pH monitoring has opened our eyes to the trafficking of much more than acid reflux through the esophageal lumen. It is clear that acid reflux as identified by a conventional pH electrode represents only a subset of reflux events with many more reflux episodes being composed of less acidic and gaseous mixtures. This has prompted many investigations into the genesis of refractory reflux symptoms. However, with both technologies, the challenge has been to make sense of the vastly expanded datasets. At the very least, HRM is a major technological tweak on conventional manometry, and impedance pH monitoring yields information above and beyond that gained from conventional pH monitoring studies. Ultimately, however, both technologies will be strengthened as outcome studies evaluating their utilization become available.     

Detection of EGFR mutation in supernatant,cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing

To determine epidermal growth factor receptor (EGFR) mutation in advanced non-small cell lung cancer (NSCLC) patients and compare the detection efficiency between different sample resources, both high resolution melting (HRM) analysis and direct sequencing method were used to analyze 36 pleural effusion samples and 22 matched biopsy tumor tissues collected from NSCLC patients. For each pleural effusion sample, the supernatant and the cell pellets were examined separately. Among all the 36 cases of pleural effusion samples, 18 mutations of EGFR were found in cell-free supernatant while 13 mutations were found in the cell pellets as detected by HRM analysis. In the 22 matched samples, 13 cases of EGFR mutations were identified in paraffin-embedded biopsy tissue samples, 12 cases in the cell-free supernatant and 9 cases in the cell pellets of pleural effusion. EGFR mutations in 15 cases out of the total 36 pleural effusion samples detected by direct sequencing were also identified by HRM analysis, giving 100% efficiency for HRM method. The results established the important role of HRM as a reliable and efficient method to determine EGFR mutation status and indicated the feasibility of using pleural effusion in replacement of biopsy tissues in particular clinical cases. Furthermore, the cell-free supernatant of pleural effusion might be a better resource for mutation detection than cell pellets.     

高分辨率熔解曲线法检测CBS基因SNP的技术优化

目的：探索高分辨率熔解曲线法（highresolutionmelting,HRM）检测胱硫醚β-合酶（CBS）基因SNP位点所需的最佳反应体系和条件,建立快速高效的对CBS基因分型的方法。方法通过普通PCR初步确立引物的退火温度范围,在实时荧光定量PCR（qPCR）-HRM反应中调整确定最佳退火温度。对影响qPCR-HRM的因素包括反应程序、模板DNA、MgCl2再分别进行优化,确立最佳反应体系和反应条件,对在此基础上得出的基因分型结果通过测序验证其准确性,从而建立系统的HRM检测CBS基因SNP的方法。结果HRM检测CBS基因该片段最佳退火温度为62℃,qPCR-HRM最佳反应体系为20μl,引物浓度为0．2μmol/L,Mg2＋为2．5μmol/L,模板DNA为60ng。在优化的体系条件下筛选出的突变型样本经测序验证与实验结果一致。结论HRM可以作为检测SNP准确、经济、高效的方法。     

精神科护士自我职业生涯规划现状调查

目的 了解精神科护士自我职业生涯规划现状,为精神科护理人力资源管理提供理论依据.方法 采用护士职业生涯规划问卷,对115名精神科护士进行调查分析.结果 本组护士自我职业生涯规划总分（38.33±5.78）分,处于中等水平;不同工龄、学历护士职业生涯规划总分比较差异均有显著性（P＜0.05）.结论 精神科护士自我职业生涯规划处于中等水平,医院应加强护士职业生涯的规划与管理,增强精神科护士自我职业生涯规划意识.     

Coinheritance of Hb A2-Melbourne (HBD: c.130G>A) and Hb E (HBB: c.79G>A) in Laos and Simultaneous High Resolution Melt Detection of Hb A2-Melbourne and Hb A2-Lampang (HBD: c.142G>A) in a Single Tube

Abstract

We report the molecular and hematological identifications of a Hb A₂ variant [coinheritance of Hb A₂-Melbourne (HBD: c.130G>A) and Hb E (HBB: c.79G>A)] found for the first time in the Lao People’s Democratic Republic (PDR). The subject was a 29-year-old pregnant Laotian woman who was a foreign worker in Thailand and was diagnosed with thalassemia and hemoglobinopathies. Capillary electrophoresis (CE) demonstrated 1.6% of Hb A₂, with a minor unknown peak at the initial Z1 zone (1.7%). Identification of abnormal hemoglobin (Hb) using direct DNA sequencing showed a genetic defect causing a δ-globin gene missense mutation at codon 43 (GAG>AAG) causing a glutamic acid to lysine substitution corresponding to Hb A₂-Melbourne. The origin of Hb A₂-Melbourne in Lao PDR may be similar to a case found in Thailand with the [+ –?– –?– + +] haplotype. We developed a method that could clearly detect Hb A₂-Melbourne and Hb A₂-Lampang (HBD: c.142G>A) mutations in a single tube using high resolution melt (HRM) analysis. The HRM analysis is a more effective method for rapid detection than conventional polymerase chain reaction (PCR), as there is no need for a post-PCR step, and no exposure to ethidium bromide. This new method would be a useful addition for the first investigation of a suspected Hb A₂ variant in the routine molecular setting.     

Expansion of urban cutaneous leishmaniasis into rural areas of southeastern Iran: Clinical,epidemiological and phylogenetic profiles explored using 7SL high resolution melting‐PCR analysis

Cutaneous leishmaniasis (CL) has increased remarkably in Iran and has expanded into new areas. The present study aimed to assess the emerging CL outbreak in southeastern Iran using high resolution melting‐polymerase chain reaction (HRM‐PCR) and phylogenetic analysis using the 7SL RNA gene marker. A cross‐sectional and analytical survey was conducted during a house‐to‐house census of 11,021 inhabitants in Narmashir County in southeastern Iran in 2016. The cases were detected by direct smear microscopic examination and sequencing and were characterized using the 7SL RNA gene. All age groups and sexes were equally affected. Most were single lesions (70.7%). The hands (55.2%) and face (37.9%) were the main sites of involvement. The disease was more common among illiterate persons. Sequencing and HRM‐PCR revealed that Leishmania tropica (accession no. MH632168 Qale‐Shahid) was the principal causative agent of anthroponotic CL (ACL) in new areas of expansion. This is the first emergence of ACL in rural areas of Narmashir County. Based on the molecular data, the causative parasite species confirmed to be L. tropica. Sequencing and phylogenetic analysis indicated that a single clone of the organism derived from a single source has spread into the affected villages. Construction of a main road, population movement and recent urbanization in the area are likely the major factors associated with the establishment of this new outbreak. This study was essential to enable the planning of effective therapeutic and prophylactic measures to control the disease.     

Sensitive High-Resolution Melting Analysis for Screening of KRAS and BRAF Mutations in Iranian Human Metastatic Colorectal Cancers

Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes.     

Increased sensitivity of KRAS mutation detection by high‐resolution melting analysis of COLD‐PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low‐level tumor specific KRAS mutations. Coamplification at lower denaturation temperature‐PCR (COLD‐PCR) is a new form of PCR that selectively amplifies mutation‐containing templates based on the lower melting temperature of mutant homoduplexes versus wild‐type homoduplexes. We have developed a fast COLD‐PCR and high‐resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD‐PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen^® KRAS mutation kit. The sensitivity was increased by 5‐ to 100‐fold for melting temperature decreasing mutations when using COLD‐PCR compared to standard PCR. Mutations, undetectable by the TheraScreen^® kit in clinical samples, were detected by COLD‐PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD‐PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.