Identification of six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay.

PubMed

Hernández, Carolina; Alvarez, Catalina; González, Camila; Ayala, Martha Stella; León, Cielo Maritza; Ramírez, Juan David

2014-11-14

Leishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species. Herein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL. This is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

[Mutation analysis for a pedigree affected with keratitis-ichthyosis-deafness syndrome].

PubMed

Li, Lulu; Li, Yuan; Lin, Wei; Zhao, Xiuli

2017-10-10

To identify mutation of GJB2 gene and provide genetic counseling for a family affected with keratitis-ichthyosis-deafness (KID) syndrome. Genomic DNA was extracted from peripheral blood samples with a standard phenol-chloroform method. PCR and Sanger sequencing were used to analyze potential mutation in the proband. Suspected mutation was verified with a PCR-high-resolution melting (PCR-HRM) method. T-clone sequencing was applied to determine the parental origin of the mutation. A heterozygous mutation, c.148G>A (p.Asp50Asn), which is located in the exon 1 of the GJB2 gene, was found in the proband. The results was confirmed by HRM analysis. Cloning sequencing suggested that the mutation was derived from the father's germline. The hot-spot mutation c.148G>A (p.Asp50Asn) in the GJB2 gene probably underlies the KID syndrome in this Chinese family. A PCR-HRM method has been established to rapidly detect common mutations associated with this disease.

Fast and reliable detection of toxic Crotalaria spectabilis Roth. in Thunbergia laurifolia Lindl. herbal products using DNA barcoding coupled with HRM analysis.

PubMed

Singtonat, Sahachat; Osathanunkul, Maslin

2015-05-30

Nowadays, medicinal plants are used as a popular alternative to synthetic drugs. Many medicinal plant products have now been commercialized throughout various markets. These products are commonly sold in processed or modified forms such as powders, dried material and capsules, making it almost impossible to accurately identify the constituent species. The herbal plant known as 'Rang Chuet' in Thai has been widely used as remedies for various ailments. However, two medicinal plants species, Thunbergia laurifolia and Crotalaria spectabilis share this name. Duo to the similarity in nomenclature, the commercial products labeled as 'Rang Chuet' could be any of them. Recently, the evidence of hepatotoxic effects linked to use of C. spectabilis were reported and is now seriously concern. There is a need to find an approach that could help with species identification of these herbal products to ensure the safety and efficacy of the herbal drug. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate T. laurifolia species. Four DNA barcodes including matK, rbcL, rpoC and trnL were selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Commercial products labeled as 'Rang Chuet' were purchased from Thai markets and authentication by HRM analyses. Melting data from the HRM assay using the designed primers showed that the two 'Rang Chuet' species could easily be distinguished from each other. The melting profiles of the all four region amplicons of each species are clearly separated in all three replicates. The method was then applied to authenticate products in powdered form. HRM curves of all ten test samples indicated that three of the tested products did not only contain the T. laurifolia species. The herbal drugs derived from different plants must be distinguished from each other even they share the same vernacular name. The Bar-HRM method developed here proved useful in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.

[Application of three heat pulse technique-based methods to determine the stem sap flow].

PubMed

Wang, Sheng; Fan, Jun

2015-08-01

It is of critical importance to acquire tree transpiration characters through sap flow methodology to understand tree water physiology, forest ecology and ecosystem water exchange. Tri-probe heat pulse sensors, which are widely utilized in soil thermal parameters and soil evaporation measurement, were applied to implement Salix matsudana sap flow density (Vs) measurements via heat-ratio method (HRM), T-Max method (T-Max) and single-probe heat pulse probe (SHPP) method, and comparative analysis was conducted with additional Grainer's thermal diffusion probes (TDP) measured results. The results showed that, it took about five weeks to reach a stable measurement stage after TPHP installation, Vs measured with three methods in the early stage after installation was 135%-220% higher than Vs in the stable measurement stage, and Vs estimated via HRM, T-Max and SHPP methods were significantly linearly correlated with Vs estimated via TDP method, with R2 of 0.93, 0.73 and 0.91, respectively, and R2 for Vs measured by SHPP and HRM reached 0.94. HRM had relatively higher precision in measuring low rates and reverse sap flow. SHPP method seemed to be very promising to measure sap flow for configuration simplicity and high measuring accuracy, whereas it couldn' t distinguish directions of flow. T-Max method had relatively higher error in sap flow measurement, and it couldn' t measure sap flow below 5 cm3 · cm(-2) · h(-1), thus this method could not be used alone, however it could measure thermal diffusivity for calculating sap flow when other methods were imposed. It was recommended to choose a proper method or a combination of several methods to measure stem sap flow, based on specific research purpose.

High resolution melting (HRM) analysis of DNA--its role and potential in food analysis.

PubMed

Druml, Barbara; Cichna-Markl, Margit

2014-09-01

DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

On the validation of cloud parametrization schemes in numerical atmospheric models with satellite data from ISCCP

NASA Astrophysics Data System (ADS)

Meinke, I.

2003-04-01

A new method is presented to validate cloud parametrization schemes in numerical atmospheric models with satellite data of scanning radiometers. This method is applied to the regional atmospheric model HRM (High Resolution Regional Model) using satellite data from ISCCP (International Satellite Cloud Climatology Project). Due to the limited reliability of former validations there has been a need for developing a new validation method: Up to now differences between simulated and measured cloud properties are mostly declared as deficiencies of the cloud parametrization scheme without further investigation. Other uncertainties connected with the model or with the measurements have not been taken into account. Therefore changes in the cloud parametrization scheme based on such kind of validations might not be realistic. The new method estimates uncertainties of the model and the measurements. Criteria for comparisons of simulated and measured data are derived to localize deficiencies in the model. For a better specification of these deficiencies simulated clouds are classified regarding their parametrization. With this classification the localized model deficiencies are allocated to a certain parametrization scheme. Applying this method to the regional model HRM the quality of forecasting cloud properties is estimated in detail. The overestimation of simulated clouds in low emissivity heights especially during the night is localized as model deficiency. This is caused by subscale cloudiness. As the simulation of subscale clouds in the regional model HRM is described by a relative humidity parametrization these deficiencies are connected with this parameterization.

Evaluation of Two Multiplex PCR-High-Resolution Melt Curve Analysis Methods for Differentiation of Campylobacter jejuni and Campylobacter coli Intraspecies.

PubMed

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Raidal, Shane; Ghorashi, Seyed Ali

2018-03-01

Campylobacter infection is a common cause of bacterial gastroenteritis in humans and remains a significant global public health issue. The capability of two multiplex PCR (mPCR)-high-resolution melt (HRM) curve analysis methods (i.e., mPCR1-HRM and mPCR2-HRM) to detect and differentiate 24 poultry isolates and three reference strains of Campylobacter jejuni and Campylobacter coli was investigated. Campylobacter jejuni and C. coli were successfully differentiated in both assays, but the differentiation power of mPCR2-HRM targeting the cadF gene was found superior to that of mPCR1-HRM targeting the gpsA gene or a hypothetical protein gene. However, higher intraspecies variation within C. coli and C. jejuni isolates was detected in mPCR1-HRM when compared with mPCR2-HRM. Both assays were rapid and required minimum interpretation skills for discrimination between and within Campylobacter species when using HRM curve analysis software.

Hybrid region merging method for segmentation of high-resolution remote sensing images

NASA Astrophysics Data System (ADS)

Zhang, Xueliang; Xiao, Pengfeng; Feng, Xuezhi; Wang, Jiangeng; Wang, Zuo

2014-12-01

Image segmentation remains a challenging problem for object-based image analysis. In this paper, a hybrid region merging (HRM) method is proposed to segment high-resolution remote sensing images. HRM integrates the advantages of global-oriented and local-oriented region merging strategies into a unified framework. The globally most-similar pair of regions is used to determine the starting point of a growing region, which provides an elegant way to avoid the problem of starting point assignment and to enhance the optimization ability for local-oriented region merging. During the region growing procedure, the merging iterations are constrained within the local vicinity, so that the segmentation is accelerated and can reflect the local context, as compared with the global-oriented method. A set of high-resolution remote sensing images is used to test the effectiveness of the HRM method, and three region-based remote sensing image segmentation methods are adopted for comparison, including the hierarchical stepwise optimization (HSWO) method, the local-mutual best region merging (LMM) method, and the multiresolution segmentation (MRS) method embedded in eCognition Developer software. Both the supervised evaluation and visual assessment show that HRM performs better than HSWO and LMM by combining both their advantages. The segmentation results of HRM and MRS are visually comparable, but HRM can describe objects as single regions better than MRS, and the supervised and unsupervised evaluation results further prove the superiority of HRM.

A comparison of PCR assays for beak and feather disease virus and high resolution melt (HRM) curve analysis of replicase associated protein and capsid genes.

PubMed

Das, Shubhagata; Sarker, Subir; Ghorashi, Seyed Ali; Forwood, Jade K; Raidal, Shane R

2016-11-01

Beak and feather disease virus (BFDV) threatens a wide range of endangered psittacine birds worldwide. In this study, we assessed a novel PCR assay and genetic screening method using high-resolution melt (HRM) curve analysis for BFDV targeting the capsid (Cap) gene (HRM-Cap) alongside conventional PCR detection as well as a PCR method that targets a much smaller fragment of the virus genome in the replicase initiator protein (Rep) gene (HRM-Rep). Limits of detection, sensitivity, specificity and discriminatory power for differentiating BFDV sequences were compared. HRM-Cap had a high positive predictive value and could readily differentiate between a reference genotype and 17 other diverse BFDV genomes with more discriminatory power (genotype confidence percentage) than HRM-Rep. Melt curve profiles generated by HRM-Cap correlated with unique DNA sequence profiles for each individual test genome. The limit of detection of HRM-Cap was lower (2×10 -5 ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2×10 -6 ng or 13 viral copies/reaction). However, when used in a diagnostic setting with 348 clinical samples there was strong agreement between HRM-Cap and conventional PCR (kappa=0.87, P<0.01, 98% specificity) and HRM-Cap demonstrated higher specificity (99.9%) than HRM-Rep (80.3%). Copyright © 2016 Elsevier B.V. All rights reserved.

Describing litho-constrained layout by a high-resolution model filter

NASA Astrophysics Data System (ADS)

Tsai, Min-Chun

2008-05-01

A novel high-resolution model (HRM) filtering technique was proposed to describe litho-constrained layouts. Litho-constrained layouts are layouts that have difficulties to pattern or are highly sensitive to process-fluctuations under current lithography technologies. HRM applies a short-wavelength (or high NA) model simulation directly on the pre-OPC, original design layout to filter out low spatial-frequency regions, and retain high spatial-frequency components which are litho-constrained. Since no OPC neither mask-synthesis steps are involved, this new technique is highly efficient in run time and can be used in design stage to detect and fix litho-constrained patterns. This method has successfully captured all the hot-spots with less than 15% overshoots on a realistic 80 mm2 full-chip M1 layout in 65nm technology node. A step by step derivation of this HRM technique is presented in this paper.

Molecular differentiation of three canine and feline hookworms in South China through HRM analysis.

PubMed

Wang, M W; Yan, X X; Hang, J X; Shi, X L; Fu, Y Q; Zhang, P; Yang, F; Pan, W D; Li, G Q

2018-02-05

To investigate the prevalence of canine and feline hookworms in South China, and to assess the risk of zoonotic hookworms to humans, one pair of primers (HRM-F/HRM-R) was designed to establish a high-resolution melting (HRM) method based on internal transcribed spacer 1 (ITS-1) rDNA for the detection of Ancylostoma ceylanicum, A. caninum and A. tubaeforme infection. The results showed that the HRM for the three hookworms produced different melting-curve profiles, where melting temperature (T m) values were 84.50°C for A. ceylanicum, 82.25°C for A. caninum and 81.73°C for A. tubaeforme, respectively. The reproducibility of intra- and inter-assay melting curves was almost perfect. The lowest concentration detected was about 5.69 ×10-4 g/μl. The HRM detection results from 18 canine and feline hookworm samples were in complete accordance with their sequencing results. The HRM method was more sensitive than the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in the detection of 98 clinical samples. It is concluded that the HRM method can differentiate between A. ceylanicum, A. caninum, A. tubaeforme and their mixed infections, which may provide important technical support for the zoonotic risk assessment and molecular epidemiological survey of canine and feline hookworms.

The Role of Work-Related Factors in the Development of Psychological Distress and Associated Mental Disorders: Differential Views of Human Resource Managers, Occupational Physicians, Primary Care Physicians and Psychotherapists in Germany.

PubMed

Junne, Florian; Michaelis, Martina; Rothermund, Eva; Stuber, Felicitas; Gündel, Harald; Zipfel, Stephan; Rieger, Monika A

2018-03-20

Objectives : This study analyses the perceived relevance of stress-dimensions in work-settings from the differential views of Human Resource Managers (HRM), Occupational Physicians (OP), Primary Care Physicians (PCP) and Psychotherapists (PT) in Germany. Methods : Cross-sectional study design, using a self-report questionnaire. Descriptive measures and explorative bivariate methods were applied for group-comparisons. Results are presented as rankings of perceived importance and as polarity profiles of contrasting views. Results: N = 627 participants completed the questionnaires (HRM: n = 172; OP: n = 133; PCP: n = 136; PT: n = 186). The stress dimensions with the highest mean ratings across all four professions were: 'social relationships in the work place' ( M = 3.55, SD = 0.62) and 'superiors´ leadership style' ( M = 3.54, SD = 0.64). Mean ratings of perceived relevance of stress dimensions differed most between HRM and the three medical professions. Conclusions : The perceived importance of work-related stress-dimensions seems to be higher in the medical disciplines (OP, PCP, PT) than in the group from the management sector (HRM). However, no fundamental disagreement on the role of work-related stress-dimensions seems to hinder e.g., intensified efforts of cooperation across sectors in tackling the "stress-pandemic" and improving the (mental) health of employees.

The Role of Work-Related Factors in the Development of Psychological Distress and Associated Mental Disorders: Differential Views of Human Resource Managers, Occupational Physicians, Primary Care Physicians and Psychotherapists in Germany

PubMed Central

Junne, Florian; Michaelis, Martina; Stuber, Felicitas; Gündel, Harald; Zipfel, Stephan

2018-01-01

Objectives: This study analyses the perceived relevance of stress-dimensions in work-settings from the differential views of Human Resource Managers (HRM), Occupational Physicians (OP), Primary Care Physicians (PCP) and Psychotherapists (PT) in Germany. Methods: Cross-sectional study design, using a self-report questionnaire. Descriptive measures and explorative bivariate methods were applied for group-comparisons. Results are presented as rankings of perceived importance and as polarity profiles of contrasting views. Results: N = 627 participants completed the questionnaires (HRM: n = 172; OP: n = 133; PCP: n = 136; PT: n = 186). The stress dimensions with the highest mean ratings across all four professions were: ‘social relationships in the work place’ (M = 3.55, SD = 0.62) and ‘superiors´ leadership style’ (M = 3.54, SD = 0.64). Mean ratings of perceived relevance of stress dimensions differed most between HRM and the three medical professions. Conclusions: The perceived importance of work-related stress-dimensions seems to be higher in the medical disciplines (OP, PCP, PT) than in the group from the management sector (HRM). However, no fundamental disagreement on the role of work-related stress-dimensions seems to hinder e.g., intensified efforts of cooperation across sectors in tackling the “stress-pandemic” and improving the (mental) health of employees. PMID:29558427

High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

PubMed

Distefano, Gaetano; Caruso, Marco; La Malfa, Stefano; Gentile, Alessandra; Wu, Shu-Biao

2012-01-01

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.

Water vapor over Europe obtained from remote sensors and compared with a hydrostatic NWP model

NASA Astrophysics Data System (ADS)

Johnsen, K.-P.; Kidder, S. Q.

Due to its high-variability water vapor is a crucial parameter in short-term numerical weather prediction. Integrated water vapor (IWV) data obtained from a network of groundbased Global Positioning System (GPS) receivers mainly over Germany and passive microwave measurements of the Advanced Microwave Sounding Unit (AMSU-A) are compared with the high-resolution regional weather forecast model HRM of the Deutscher Wetterdienst (DWD). Time series of the IWV at 74 GPS stations obtained during the first complete year of the GFZ/GPS network between May 2000 and April 2001 are applied together with colocated forecasts of the HRM model. The low bias (0.08 kg/m 2) between the HRM model and the GPS data can mainly be explained by the bias between the ECMWF analysis data used to initilize the HRM model and the GPS data. The IWV standard deviation between the HRM model and the GPS data during that time is about 2.47 kg/ m2. GPS stations equipped with surface pressure sensors show about 0.29 kg/ m2 lower standard deviation compared with GPS stations with interpolated surface pressure from synoptic stations. The NOAA/NESDIS Total Precipitable Water algorithm is applied to obtain the IWV and to validate the model above the sea. While the mean IWV obtained from the HRM model is about 2.1 kg/ m2 larger than from the AMSU-A data, the standard deviations are 2.46 kg/ m2 (NOAA-15) and 2.29 kg/ m2 (NOAA-16) similar to the IWV standard deviation between HRM and GPS data.

Authenticity analyses of Rhizoma Paridis using barcoding coupled with high resolution melting (Bar-HRM) analysis to control its quality for medicinal plant product.

PubMed

Duan, Bao-Zhong; Wang, Ya-Ping; Fang, Hai-Lan; Xiong, Chao; Li, Xi-Wen; Wang, Ping; Chen, Shi-Lin

2018-01-01

Rhizoma Paridis (Chonglou) is a commonly used and precious traditional Chinese medicine. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz. and Paris polyphylla Smith var . chinensis (Franch.) Hara are the two main sources of Chonglou under the monograph of Rhizoma Paridis in Chinese Pharmacopoeia. In the local marketplace, however, this medicine is prone to be accidentally contaminated, deliberately substituted or admixed with other species that are similar to Rhizoma Paridis in shape and color. Consequently, these adulterations might compromise quality control and result in considerable health concerns for consumers. This study aims to develop a rapid and sensitive method for accurate identification of Rhizoma Paridis and its common adulterants. DNA barcoding coupled with high resolution melting analysis was applied in this research to distinguish Rhizoma Paridis from its adulteration. The internal transcribed spacer 2 (ITS2) barcode was selected for HRM analysis to produce standard melting profile of the selected species. DNA of the tested herbal medicines was isolated and their melting profiles were generated and compared with the standard melting profile of P. polyphylla var. chinensis . The results indicate that the ITS2 molecular regions coupled with HRM analysis can effectively differentiate nine herbal species, including two authentic origins of Chonglou and their seven common adulterants. Ten herbal medicines labeled "Chonglou" obtained from a local market were collected and identified with our methods, and their sequence information was analyzed to validate the accuracy of HRM analysis. DNA barcoding coupled with HRM analysis is a accurate, reliable, rapid, cost-effective and robust tool, which could contribute to the quality control of Rhizoma Paridis in the supply chain of the natural health product industry (NHP).

High-resolution melting (HRM) assay for the detection of recurrent BRCA1/BRCA2 germline mutations in Tunisian breast/ovarian cancer families.

PubMed

Riahi, Aouatef; Kharrat, Maher; Lariani, Imen; Chaabouni-Bouhamed, Habiba

2014-12-01

Germline deleterious mutations in the BRCA1/BRCA2 genes are associated with an increased risk for the development of breast and ovarian cancer. Given the large size of these genes the detection of such mutations represents a considerable technical challenge. Therefore, the development of cost-effective and rapid methods to identify these mutations became a necessity. High resolution melting analysis (HRM) is a rapid and efficient technique extensively employed as high-throughput mutation scanning method. The purpose of our study was to assess the specificity and sensitivity of HRM for BRCA1 and BRCA2 genes scanning. As a first step we estimate the ability of HRM for detection mutations in a set of 21 heterozygous samples harboring 8 different known BRCA1/BRCA2 variations, all samples had been preliminarily investigated by direct sequencing, and then we performed a blinded analysis by HRM in a set of 68 further sporadic samples of unknown genotype. All tested heterozygous BRCA1/BRCA2 variants were easily identified. However the HRM assay revealed further alteration that we initially had not searched (one unclassified variant). Furthermore, sequencing confirmed all the HRM detected mutations in the set of unknown samples, including homozygous changes, indicating that in this cohort, with the optimized assays, the mutations detections sensitivity and specificity were 100 %. HRM is a simple, rapid and efficient scanning method for known and unknown BRCA1/BRCA2 germline mutations. Consequently the method will allow for the economical screening of recurrent mutations in Tunisian population.

Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

PubMed

Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing

2015-11-01

Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia.

Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

PubMed Central

Do, Hongdo; Dobrovic, Alexander

2009-01-01

Background Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations. We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Results Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions. LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. Conclusion LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations. PMID:19811662

Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

PubMed

Do, Hongdo; Dobrovic, Alexander

2009-10-08

Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

PubMed

Macher, Hada C; Martinez-Broca, Maria A; Rubio-Calvo, Amalia; Leon-Garcia, Cristina; Conde-Sanchez, Manuel; Costa, Alzenira; Navarro, Elena; Guerrero, Juan M

2012-01-01

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection

PubMed Central

Cousins, Matthew M.; Laeyendecker, Oliver; Beauchamp, Geetha; Brookmeyer, Ronald; Towler, William I.; Hudelson, Sarah E.; Khaki, Leila; Koblin, Beryl; Chesney, Margaret; Moore, Richard D.; Kelen, Gabor D.; Coates, Thomas; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

2011-01-01

Background Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. Methods HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Results Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. Conclusions The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined. PMID:22073290

High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

PubMed

Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

2016-07-01

Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.

Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

PubMed

Solano, Jaime; Anabalón, Leonardo; Encina, Francisco

2016-03-01

Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study.

PubMed

Libert, Xavier; Packeu, Ann; Bureau, Fabrice; Roosens, Nancy H; De Keersmaecker, Sigrid C J

2017-04-04

Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR®Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii. In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii. Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples. We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems.

DNA barcoding coupled to HRM analysis as a new and simple tool for the authentication of Gadidae fish species.

PubMed

Fernandes, Telmo J R; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

2017-09-01

This work aimed to exploit the use of DNA mini-barcodes combined with high resolution melting (HRM) for the authentication of gadoid species: Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Theragra chalcogramma) and saithe (Pollachius virens). Two DNA barcode regions, namely cytochrome c oxidase subunit I (COI) and cytochrome b (cytb), were analysed in silico to identify genetic variability among the four species and used, subsequently, to develop a real-time PCR method coupled with HRM analysis. The cytb mini-barcode enabled best discrimination of the target species with a high level of confidence (99.3%). The approach was applied successfully to identify gadoid species in 30 fish-containing foods, 30% of which were not as declared on the label. Herein, a novel approach for rapid, simple and cost-effective discrimination/clustering, as a tool to authenticate Gadidae fish species, according to their genetic relationship, is proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

The spa typing of methicillin-resistant Staphylococcus aureus isolates by High Resolution Melting (HRM) analysis.

PubMed

Fasihi, Yasser; Fooladi, Saba; Mohammadi, Mohammad Ali; Emaneini, Mohammad; Kalantar-Neyestanaki, Davood

2017-09-06

Molecular typing is an important tool for control and prevention of infection. A suitable molecular typing method for epidemiological investigation must be easy to perform, highly reproducible, inexpensive, rapid and easy to interpret. In this study, two molecular typing methods including the conventional PCR-sequencing method and high resolution melting (HRM) analysis were used for staphylococcal protein A (spa) typing of 30 Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from clinical samples. Based on PCR-sequencing method results, 16 different spa types were identified among the 30 MRSA isolates. Among the 16 different spa types, 14 spa types separated by HRM method. Two spa types including t4718 and t2894 were not separated from each other. According to our results, spa typing based on HRM analysis method is very rapid, easy to perform and cost-effective, but this method must be standardized for different regions, spa types, and real-time machinery.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

PubMed

Bartošík, M; Ondroušková, E

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Methylation-Sensitive High Resolution Melting (MS-HRM).

PubMed

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

Systematic review of the links between human resource management practices and performance.

PubMed

Patterson, M; Rick, J; Wood, S; Carroll, C; Balain, S; Booth, A

2010-10-01

In recent years human resource management (HRM) has been seen as an important factor in the successful realisation of organisational change programmes. The UK NHS is undergoing substantial organisational change and there is a need to establish which human resource (HR) initiatives may be most effective. To assess the results from a wide-ranging series of systematic reviews of the evidence on HRM and performance. The first part assesses evidence on use of HRM in the UK and fidelity of practice implemented. The second part considers evidence for the impact of HRM practices on intermediate outcomes, which can impact on final outcomes, such as organisational performance or patient care. The following databases were searched: Applied Social Sciences Index and Abstracts (ASSIA), British Nursing Index (BNI), Business Source Premier, Campbell Collaboration, Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Database of Systematic Reviews (CDSR), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Database of Abstracts of Reviews of Effectiveness (DARE), DH-Data, EMBASE, Health Management Information Consortium (HMIC), International Bibliography of the Social Sciences (IBSS), King's Fund database, MEDLINE, NHS Economic Evaluation Database (NHS EED), National Research Register (NRR), PREMEDLINE, PsycINFO, ReFeR, Social Sciences Citation Index (SSCI) and Science Citation Index (SCI). The searches were conducted in May/June 2006. Broad categories of HRM interventions and intermediate outcomes were generated: 10 HRM categories and 12 intermediate outcome categories. Seven patient final outcomes were derived from the NHS Performance Indicators and the NHS Improvement Plan. The quality criteria used to select papers incorporated a longitudinal study design filter to provide evidence of the causal direction of relationships between HRM and relevant outcomes. Single HRM practices were considered. Within the health-specific literature, focus was on the impact of HRM on patient outcomes. Information is presented on the reliability of measures in each of the intermediate outcome areas. Work design practices that enhance employee autonomy and control influenced a number of outcomes and there was consistent evidence for the positive impact of increased job control on employee outcomes, such as job satisfaction, absence and health. For employee participation, the small number of studies reviewed supported the involvement of employees in design/implementation of changes that affect their work. In health literature in particular, employee involvement through quality improvement teams resulted in improved patient outcomes. Findings were positive for the impact of training on the intended outcomes of the initiatives. Support for the impact of performance management practices was apparent, in particular feedback on performance outcomes and the use of participative goal setting. Strong associations were found among all intermediate outcomes, and the relationship between most intermediate behaviours and outcomes were significant. Limited evidence was available on the use of HRM and on the implementation of policy. Also, the specific practices studied within each HRM category differ so there was little evidence to show whether similar practices have the same effects in health and non-health settings. Some potentially effective practices for both health and non-health areas were identified, and HRM methods could be used to support change processes within the NHS; the findings relating to work organisation are particularly promising with regard to changes in methods of service delivery. Using training to support the implementation of change is highlighted. However, future multilevel studies that embrace the individual, team and organisational level are needed. Studies should look into interventions aimed at improving HR outcomes and performance, and allow for pre- and post-intervention measurement of practices and outcomes.

Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

PubMed

Hoseinpour, Fatemeh; Foroughi, Azadeh; Nomanpour, Bizhan; Nasab, Rezvan Sobhani

2017-07-01

Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C. jejuni and C. coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C. jejuni and C. coli. A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. Fifty-five of 100 samples were positive as campylobacter (7 C. jejuni, 29 C. coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C. coli or C. jejuni isolates and this variation can be used to the differentiation between C. coli or C. jejuni isolates. The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C. jejuni and C. coli isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alternative SNP detection platforms, HRM and biosensors, for varietal identification in Vitis vinifera L. using F3H and LDOX genes.

PubMed

Gomes, Sónia; Castro, Cláudia; Barrias, Sara; Pereira, Leonor; Jorge, Pedro; Fernandes, José R; Martins-Lopes, Paula

2018-04-11

The wine sector requires quick and reliable methods for Vitis vinifera L. varietal identification. The number of V. vinifera varieties is estimated in about 5,000 worldwide. Single Nucleotide Polymorphisms (SNPs) represent the most basic and abundant form of genetic sequence variation, being adequate for varietal discrimination. The aim of this work was to develop DNA-based assays suitable to detect SNP variation in V. vinifera, allowing varietal discrimination. Genotyping by sequencing allowed the detection of eleven SNPs on two genes of the anthocyanin pathway, the flavanone 3-hydroxylase (F3H, EC: 1.14.11.9), and the leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19; synonym anthocyanidin synthase, ANS) in twenty V. vinifera varieties. Three High Resolution Melting (HRM) assays were designed based on the sequencing information, discriminating five of the 20 varieties: Alicante Bouschet, Donzelinho Tinto, Merlot, Moscatel Galego and Tinta Roriz. Sanger sequencing of the HRM assay products confirmed the HRM profiles. Three probes, with different lengths and sequences, were used as bio-recognition elements in an optical biosensor platform based on a long period grating (LPG) fiber optic sensor. The label free platform detected a difference of a single SNP using genomic DNA samples. The two different platforms were successfully applied for grapevine varietal identification.

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification

PubMed Central

Sun, Wei; Li, Jing-jian; Xiong, Chao; Zhao, Bo; Chen, Shi-lin

2016-01-01

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines. PMID:27066026

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification.

PubMed

Sun, Wei; Li, Jing-Jian; Xiong, Chao; Zhao, Bo; Chen, Shi-Lin

2016-01-01

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines.

Conditions of High Resolution Melting Analysis on the Cobas z480 Instrument for the Genotyping of VKORC1 in the Clinical Routine Laboratory.

PubMed

Paar, Christian; Hammerl, Verena; Blessberger, Hermann; Stekel, Herbert; Steinwender, Clemens; Berg, Jörg

2016-12-01

High resolution melting (HRM) of amplicons is a simple method for genotyping of single nucleotide polymorphisms (SNPs). Albeit many applications reported, HRM seems to be rarely used in clinical laboratories. The suitability of HRM-PCR for the clinical laboratory was investigated for genotyping of SNPs of the vitamin K epoxide reductase complex unit 1 gene. About 100 DNA samples were analyzed by two different HRM-PCRs on the Cobas z480 instrument and compared with a PCR with fluorescently labeled probes (HybProbe-PCR) on the LightCycler 2.0 instrument as reference. Reliable genotyping with 100% matching results was obtained, when the amplicon size was small (63 bp) and DNA input was limited by e.g., sample dilution with salt-free water. DNA extracted by differing methods may be used for genotyping by HRM-PCR. Compared with HybProbe-PCR, HRM-PCR on the Cobas z480 instrument allows for higher through-put, however, at the cost of a higher degree of laboratory standardization and a slower turnaround.

Greek PDO saffron authentication studies using species specific molecular markers.

PubMed

Bosmali, I; Ordoudi, S A; Tsimidou, M Z; Madesis, P

2017-10-01

Saffron, the spice produced from the red stigmas of the flower of Crocus sativus L. is a frequent target of fraud and mislabeling practices that cannot be fully traced using the ISO 3632 trade standard specifications and test methods. A molecular approach is proposed herein as a promising branding strategy for the authentication of highly esteemed saffron brands such as the Greek Protected Designation of Origin (PDO) "Krokos Kozanis". Specific ISSR (inter-simple sequence repeat) markers were used to assess for the first time, the within species variability of several populations of C. sativus L. from the cultivation area of "Krokos Kozanis" as well as the potential differences with the band pattern produced by other Crocus species. Then, species-specific markers were developed taking advantage of an advanced molecular technique such as the HRM analysis coupled with universal DNA barcoding regions (trnL) (Bar-HRM) and applied to saffron admixtures with some of the most common plant adulterants (Calendula officinalis, Carthamus tinctorius, Gardenia jasminoides, Zea mays and Curcuma longa). The sensitivity of the procedure was tested for turmeric as a case study whereas HPLC-fluorescence determination of secondary metabolites was also employed for comparison. The overall results indicated that the Bar-HRM approach is quite effective in terms of specificity and sensitivity. Its effectiveness regarding the detection of turmeric was comparable to that of a conventional HPLC method (0.5% vs 1.0%, w/w). Yet, the proposed DNA-based method is much faster, cost-effective and can be used even by non-geneticists, in any laboratory having access to an HRM-capable real-time PCR instrumentation. It can be, thus, regarded as a strong analytical tool in saffron authentication studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Genetic polymorphisms of ARL15 and HLA-DMA are associated with rheumatoid arthritis in Han population from northwest China].

PubMed

Wang, Jiao; Qi, Xiaoming; Zhang, Xiaozhen; Yan, Wen; You, Chongge

2017-12-01

Objective To establish the methods for detecting single nucleotide polymorphisms (SNPs) of ADP-ribosylation factor-like GTPase 15 (ARL15), major histocompatibility complex class II-DM alpha (HLA-DMA ) and nuclear factor kappa B subunit 2 (NFKB2) genes using high resolution melting (HRM) technology, and to explore the association of those SNPs with the susceptibility of rheumatoid arthritis (RA) in northwestern Han Chinese population. Methods The PCR-HRM detection system for four SNPs (rs255758, rs1063478, rs397514331 and rs397514332) was established for genotyping, and gene sequencing was performed to validate the genotyping ability of the system. 588 RA cases and 200 controls were enrolled in a case-control study to analyze the associations of ARL15 and HLA-DMA gene polymorphisms with RA risk. Results The direct sequencing validated that the established PCR-HRM detection system could be used for genotyping clinical samples correctly. The mutated genotype of rs397514331 and rs397514332 from NFKB2 gene are not found in this study. The genotype frequencies of rs255758 and rs1063478 had statistical difference between the cases and controls, but no statistical difference in allelic frequencies. Under the dominant model (AA vs AC/CC), the AA genotype of rs255758 decreases the RA risk (OR=0.666, 95%CI=0.478-0.927, P=0.016). Conclusion The method of PCR-HRM we established can be applied to the routine detection of rs255758, rs1063478, rs397514331 and rs397514332. The ARL15 and HLA-DMA gene polymorphisms are associated with RA risk in Northwestern Han Chinese population.

High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

PubMed Central

Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

2009-01-01

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

Use of a high resolution melting (HRM) assay to compare gag, pol, and env diversity in adults with different stages of HIV infection.

PubMed

Cousins, Matthew M; Laeyendecker, Oliver; Beauchamp, Geetha; Brookmeyer, Ronald; Towler, William I; Hudelson, Sarah E; Khaki, Leila; Koblin, Beryl; Chesney, Margaret; Moore, Richard D; Kelen, Gabor D; Coates, Thomas; Celum, Connie; Buchbinder, Susan P; Seage, George R; Quinn, Thomas C; Donnell, Deborah; Eshleman, Susan H

2011-01-01

Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14-540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.

A European-wide 222radon and 222radon progeny comparison study

NASA Astrophysics Data System (ADS)

Schmithüsen, Dominik; Chambers, Scott; Fischer, Bernd; Gilge, Stefan; Hatakka, Juha; Kazan, Victor; Neubert, Rolf; Paatero, Jussi; Ramonet, Michel; Schlosser, Clemens; Schmid, Sabine; Vermeulen, Alex; Levin, Ingeborg

2017-04-01

Although atmospheric 222radon (222Rn) activity concentration measurements are currently performed worldwide, they are being made by many different laboratories and with fundamentally different measurement principles, so compatibility issues can limit their utility for regional-to-global applications. Consequently, we conducted a European-wide 222Rn / 222Rn progeny comparison study in order to evaluate the different measurement systems in use, determine potential systematic biases between them, and estimate correction factors that could be applied to harmonize data for their use as a tracer in atmospheric applications. Two compact portable Heidelberg radon monitors (HRM) were moved around to run for at least 1 month at each of the nine European measurement stations included in this comparison. Linear regressions between parallel data sets were calculated, yielding correction factors relative to the HRM ranging from 0.68 to 1.45. A calibration bias between ANSTO (Australian Nuclear Science and Technology Organisation) two-filter radon monitors and the HRM of ANSTO / HRM = 1.11 ± 0.05 was found. Moreover, for the continental stations using one-filter systems that derive atmospheric 222Rn activity concentrations from measured atmospheric progeny activity concentrations, preliminary 214Po / 222Rn disequilibrium values were also estimated. Mean station-specific disequilibrium values between 0.8 at mountain sites (e.g. Schauinsland) and 0.9 at non-mountain sites for sampling heights around 20 to 30 m above ground level were determined. The respective corrections for calibration biases and disequilibrium derived in this study need to be applied to obtain a compatible European atmospheric 222Rn data set for use in quantitative applications, such as regional model intercomparison and validation or trace gas flux estimates with the radon tracer method.

La gestion des gestionnaires de projets: Le cas de l'industrie aerospatiale au Quebec

NASA Astrophysics Data System (ADS)

Petit, Marie-Claude

This research focuses on the management of project managers (PMs) working in high technology organizations. In particular, it examines how these professionals are taken charge of from a human resources management (HRM) standpoint in firms within the aerospace industry in Quebec. The literature in the field of project management generally depicts PMs as entrepreneurs and resource managers, managing human resources (e.g. project team members) among other things. It also defines them as single-handedly responsible for managing their own career, stress and motivation. This profile suggests that once selected and assigned to projects, PMs receive little attention in terms of HRM from the organization. It is hardly conceivable, however, that PMs would move about completely on their own within the organization, let alone that they wouldn't represent any special challenge in terms of HRM, as the ones the literature so clearly underlines for project team members. The review of the literature also reveals that the subject of HRM as applied to PMs is poorly documented empirically, especially on the topic of what particular challenges the project context might bring up for the HRM of PMs. There is also little told about the actors involved in supporting and contributing to meet the challenges that arise out of managing PMs. This study sought to discern those challenges and the means put forth to address them. Also, it wanted to identify the actors of the HRM of PMs, their roles, their skills, and their relationships, among themselves and with the PMs, in order to efficiently take charge of them. This study also wanted to demonstrate the logic that ties the means put forth by the identified actors with the objectives PM-employing organizations want to reach. Finally, the study wanted to identify the impact of the HRM of PMs as it is occurring in organizations. The Quebec aerospace sector was chosen as the field of research primarily because the success of projects in organizations of that sector, alongside regular manufacturing operations, seems to contribute to their performance. In fact, most of these organizations currently are industry leaders worldwide. The data that form the basis of this research were obtained through 65 semi-structured interviews with representatives of project management, HRM and upper management in eight organizations in the aerospace industry in Quebec. First, the collected data was treated case by case using the qualitative data analysis software application Atlas.ti. Subsequently, the description and interpretation of the data were developed in a transversal way to achieve a sector-wide portrait of the HRM of PMs. The results reveal that PMs, like other human resources, do present particular HRM challenges and are actually managed as such in the organizations. PMs benefit both from traditional HRM practices (collective HRM) and from the relationship established and maintained with their immediate manager (individualized HRM). A network of actors is also involved in the HRM of PMs. Among these, the immediate manager of PMs stands out as the key HRM actor. In addition to applying HRM policies and practices to PMs, the manager of PMs, in his position of proximity with PMs, is destined to play different roles such as coach, motivator and talent agent. This hybrid HRM of PMs provides a sense of well-being among PMs and allows organizations to retain these creators of value, it secures faithful customers (repeat business) as a consequence of the success of projects managed by those PMs, and contributes to build a pool for their future managerial elite. Keywords: project management, human resource management, project manager, proximity management, high technology, aerospace industry, Quebec.

Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees.

PubMed

Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

2016-01-01

Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily determine the A. paniculata species in herbal products tested. Abbreviations used: bp: Base pair, Tm: Melting temperature.

High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.

PubMed

Tamburro, M; Ripabelli, G

2017-01-01

Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools. To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health. A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available. HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations. Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.

Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

PubMed Central

Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

2012-01-01

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

PubMed Central

Pietzka, Ariane T.; Stöger, Anna; Huhulescu, Steliana; Allerberger, Franz; Ruppitsch, Werner

2011-01-01

The ability to accurately track Listeria monocytogenes strains involved in outbreaks is essential for control and prevention of listeriosis. Because current typing techniques are time-consuming, cost-intensive, technically demanding, and difficult to standardize, we developed a rapid and cost-effective method for typing of L. monocytogenes. In all, 172 clinical L. monocytogenes isolates and 20 isolates from culture collections were typed by high-resolution melting (HRM) curve analysis of a specific locus of the internalin B gene (inlB). All obtained HRM curve profiles were verified by sequence analysis. The 192 tested L. monocytogenes isolates yielded 15 specific HRM curve profiles. Sequence analysis revealed that these 15 HRM curve profiles correspond to 18 distinct inlB sequence types. The HRM curve profiles obtained correlated with the five phylogenetic groups I.1, I.2, II.1, II.2, and III. Thus, HRM curve analysis constitutes an inexpensive assay and represents an improvement in typing relative to classical serotyping or multiplex PCR typing protocols. This method provides a rapid and powerful screening tool for simultaneous preliminary typing of up to 384 samples in approximately 2 hours. PMID:21227395

A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

PubMed

Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

2017-10-01

Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

HRM and SNaPshot as alternative forensic SNP genotyping methods.

PubMed

Mehta, Bhavik; Daniel, Runa; McNevin, Dennis

2017-09-01

Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

[High resolution melting analysis for detecting of JAK2V617F mutation in patients with myeloproliferative neoplasms].

PubMed

Chen, Hai-Hua; Yang, Ji-Long; Lu, Hui-Fang; Zhou, Wei-Jun; Yao, Fei; Deng, Lan

2014-02-01

This study was purposed to investigate the feasibility of high resolution melting (HRM) in the detection of JAK2V617F mutation in patients with myeloproliferative neoplasm (MPN). The 29 marrow samples randomly selected from patients with clinically diagnosed MPN from January 2008 to January 2011 were detected by HRM method. The results of HRM analysis were compared with that detected by allele specific polymerase chain reaction (AS-PCR) and DNA direct sequencing. The results showed that the JAK2V617F mutations were detected in 11 (37.9%, 11/29) cases by HRM, and its comparability with the direct sequencing result was 100%. While the consistency of AS-PCR with the direct sequencing was moderate (Kappa = 0.179, P = 0.316). It is concluded that the HRM analysis may be an optimal method for clinical screening of JAK2V617F mutation due to its simplicity and promptness with a high specificity.

Ability of High-Resolution Manometry to Determine Feeding Method and to Predict Aspiration Pneumonia in Patients With Dysphagia.

PubMed

Park, Chul-Hyun; Lee, Yong-Taek; Yi, Youbin; Lee, Jung-Sang; Park, Jung Ho; Yoon, Kyung Jae

2017-07-01

The introduction of high-resolution manometry (HRM) offered an improved method to objectively analyze the status of pharynx and esophagus. At present, HRM for patients with oropharyngeal dysphagia has been poorly studied. We aimed to determine feeding method and predict the development of aspiration pneumonia in patients with oropharyngeal dysphagia using HRM. We recruited 120 patients with dysphagia who underwent both HRM and videofluoroscopic swallow study. HRM was used to estimate pressure events from velopharynx (VP) to upper esophageal sphincter (UES). Feeding methods were determined to non-oral or oral feeding according to dysphagia severity. We prospectively followed patients to assess the development of aspiration pneumonia. VP maximal pressure and UES relaxation duration were independently associated with non-oral feeding. Non-oral feeding was determined based on optimal cutoff value of 105.0 mm Hg for VP maximal pressure (95.0% sensitivity and 70.0% specificity) and 0.45 s for UES relaxation duration (76.3% sensitivity and 57.5% specificity), respectively. During a mean follow-up of 18.8 months, 15.8% of patients developed aspiration pneumonia. On multivariate Cox regression analysis, VP maximal pressure (P<0.01) and UES relaxation duration (P<0.05) independently predicted the development of aspiration pneumonia. Cumulative incidence of aspiration pneumonia was significantly increased in patients with readings below optimal cutoff values for VP maximal pressure (P<0.01) and UES relaxation duration (P<0.01), individually. We first established the optimal thresholds for HRM parameters to determine feeding method and predict the development of aspiration pneumonia in patients with oropharyngeal dysphagia.

[GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].

PubMed

Skorodumova, L O; Babalyan, K A; Sultanov, R; Vasiliev, A O; Govorov, A V; Pushkar, D Y; Prilepskaya, E A; Danilenko, S A; Generozov, E V; Larin, A K; Kostryukova, E S; Sharova, E I

2016-11-01

There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450). However, the consistency of MS-HRM results with chip hybridization results has not been examined yet. The aim of this study was to assess the consistency of results of GSTP1, APC and RASSF1 gene methylation analysis in ÐÑ biopsy samples obtained by MS-HRM and chip hybridization. The methylation levels of each gene determined by MS-HRM were statistically different in the group of PC tissue samples and the samples without signs of tumor growth. Chip hybridization data analysis confirmed the results obtained with the MS-HRM. Differences in methylation levels between tumor tissue and histologically intact tissue of each sample determined by MS-HRM and chip hybridization, were consistent with each other. Thus, we showed that the assessment of GSTP1, APC and RASSF1 gene methylation analysis using MS-HRM is suitable for the design of laboratory assays that will differentiate the PC tissue from the tissue without signs of tumor growth.

Identification of Uvaria sp by barcoding coupled with high-resolution melting analysis (Bar-HRM).

PubMed

Osathanunkul, M; Madesis, P; Ounjai, S; Pumiputavon, K; Somboonchai, R; Lithanatudom, P; Chaowasku, T; Wipasa, J; Suwannapoom, C

2016-01-13

DNA barcoding, which was developed about a decade ago, relies on short, standardized regions of the genome to identify plant and animal species. This method can be used to not only identify known species but also to discover novel ones. Numerous sequences are stored in online databases worldwide. One of the ways to save cost and time (by omitting the sequencing step) in species identification is to use available barcode data to design optimized primers for further analysis, such as high-resolution melting analysis (HRM). This study aimed to determine the effectiveness of the hybrid method Bar-HRM (DNA barcoding combined with HRM) to identify species that share similar external morphological features, rather than conduct traditional taxonomic identification that require major parts (leaf, flower, fruit) of the specimens. The specimens used for testing were those, which could not be identified at the species level and could either be Uvaria longipes or Uvaria wrayias, indicated by morphological identification. Primer pairs derived from chloroplast regions (matK, psbA-trnH, rbcL, and trnL) were used in the Bar-HRM. The results obtained from psbA-trnH primers were good enough to help in identifying the specimen while the rest were not. Bar-HRM analysis was proven to be a fast and cost-effective method for plant species identification.

Developing and Evaluating the HRM Technique for Identifying Cytochrome P450 2D6 Polymorphisms.

PubMed

Lu, Hsiu-Chin; Chang, Ya-Sian; Chang, Chun-Chi; Lin, Ching-Hsiung; Chang, Jan-Gowth

2015-05-01

Cytochrome P450 2D6 is one of the important enzymes involved in the metabolism of many widely used drugs. Genetic polymorphisms of CYP2D6 can affect its activity. Therefore, an efficient method for identifying CYP2D6 polymorphisms is clinically important. We developed a high-resolution melting (HRM) analysis to investigate CYP2D6 polymorphisms. Genomic DNA was extracted from peripheral blood samples from 71 healthy individuals. All nine exons of the CYP2D6 gene were sequenced before screening by HRM analysis. This method can detect the most genotypes (*1, *2, *4, *10, *14, *21 *39, and *41) of CYP2D6 in Chinese. All samples were successfully genotyped. The four most common mutant CYP2D6 alleles (*1, *2, *10, and *41) can be genotyped. The single nucleotides polymorphism (SNP) frequencies of 100C > T (rs1065852), 1039C > T (rs1081003), 1661G > C (rs1058164), 2663G > A (rs28371722), 2850C > T (rs16947), 2988G > A (rs28371725), 3181A > G, and 4180G > C (rs1135840) were 58%, 61%, 73%, 1%, 13%, 3%, 1%, 73%, respectively. We identified 100% of all heterozygotes without any errors. The two homozygous genotypes (1661G > C and 4180G > C) can be distinguished by mixing with a known genotype sample to generate an artificial heterozygote for HRM analysis. Therefore, all samples could be identified using our HRM method, and the results of HRM analysis are identical to those obtained by sequencing. Our method achieved 100% sensitivity, specificity, positive prediction value and negative prediction value. HRM analysis is a nongel resolution method that is faster and less expensive than direct sequencing. Our study shows that it is an efficient tool for typing CYP2D6 polymorphisms. © 2014 Wiley Periodicals, Inc.

Health worker motivation in Africa: the role of non-financial incentives and human resource management tools

PubMed Central

Mathauer, Inke; Imhoff, Ingo

2006-01-01

Background There is a serious human resource crisis in the health sector in developing countries, particularly in Africa. One of the challenges is the low motivation of health workers. Experience and the evidence suggest that any comprehensive strategy to maximize health worker motivation in a developing country context has to involve a mix of financial and non-financial incentives. This study assesses the role of non-financial incentives for motivation in two cases, in Benin and Kenya. Methods The study design entailed semi-structured qualitative interviews with doctors and nurses from public, private and NGO facilities in rural areas. The selection of health professionals was the result of a layered sampling process. In Benin 62 interviews with health professionals were carried out; in Kenya 37 were obtained. Results from individual interviews were backed up with information from focus group discussions. For further contextual information, interviews with civil servants in the Ministry of Health and at the district level were carried out. The interview material was coded and quantitative data was analysed with SPSS software. Results and discussion The study shows that health workers overall are strongly guided by their professional conscience and similar aspects related to professional ethos. In fact, many health workers are demotivated and frustrated precisely because they are unable to satisfy their professional conscience and impeded in pursuing their vocation due to lack of means and supplies and due to inadequate or inappropriately applied human resources management (HRM) tools. The paper also indicates that even some HRM tools that are applied may adversely affect the motivation of health workers. Conclusion The findings confirm the starting hypothesis that non-financial incentives and HRM tools play an important role with respect to increasing motivation of health professionals. Adequate HRM tools can uphold and strengthen the professional ethos of doctors and nurses. This entails acknowledging their professionalism and addressing professional goals such as recognition, career development and further qualification. It must be the aim of human resources management/quality management (HRM/QM) to develop the work environment so that health workers are enabled to meet their personal and the organizational goals. PMID:16939644

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

PubMed

Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

High-resolution manometry classifications for idiopathic achalasia in patients with Chagas' disease esophagopathy.

PubMed

Vicentine, Fernando P P; Herbella, Fernando A M; Allaix, Marco E; Silva, Luciana C; Patti, Marco G

2014-02-01

Idiopathic achalasia (IA) and Chagas' disease esophagopathy (CDE) share several similarities. The comparison between IA and CDE is important to evaluate whether treatment options and their results can be accepted universally. High-resolution manometry (HRM) has proved a better diagnostic tool compared to conventional manometry. This study aims to evaluate HRM classifications for idiopathic achalasia in patients with CDE. We studied 98 patients: 52 patients with CDE (52 % females, mean age, 57 ± 14 years) and 46 patients with IA (54 % females; mean age 48 ± 19 years). All patients underwent a HRM and barium esophagogram. The Chicago classification was distributed in IA as Chicago I, 35 %; Chicago II, 63 %; and Chicago III, 2 %, and in CDE as Chicago I, 52 %; Chicago II, 48 %; and Chicago III, 0 % (p = 0.1, 0.1, and 0.5, respectively). All patients had the classic Rochester type. CDE patients had more pronounced degrees of esophageal dilatation (p < 0.002). The degree of esophageal dilatation did not correlate with Chicago classification (p = 0.08). In nine (9 %) patients, the HRM pattern changed during the test from Chicago I to II. Our results show that (a) HRM classifications for IA can be applied in patients with CDE and (b) HRM classifications did not correlate with the degree of esophageal dilatation. HRM classifications may reflect esophageal repletion and pressurization instead of muscular contraction. The correlation between manometric findings and treatment outcomes for CDE needs to be answered in the near future.

High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers.

PubMed

Zafari, Mandana; Gill, Pooria; Kowsaryan, Mehrnoush; Alipour, Abbass; Banihashemi, Ali

2016-10-01

The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of β-thalassemia. Genomic DNAs were prepared from 50 β-thalassemia minor couples whose pregnancy was at risk for homozygous β-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test. The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively. HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs.

PubMed

Mishra, Priyanka; Shukla, Ashutosh K; Sundaresan, Velusamy

2018-01-01

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes ( rbcL and matK ) and intergenic spacers ( psbA-trnH and ITS ) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S . italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 ( S. alexandrina crude drug sample from Bangalore) and HSA06 ( S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S . italica subsp. micrantha . Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs

PubMed Central

Mishra, Priyanka; Shukla, Ashutosh K.; Sundaresan, Velusamy

2018-01-01

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes (rbcL and matK) and intergenic spacers (psbA-trnH and ITS) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S. italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 (S. alexandrina crude drug sample from Bangalore) and HSA06 (S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S. italica subsp. micrantha. Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31 PMID:29593755

High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand.

PubMed

Charoenkwan, Pimlak; Sirichotiyakul, Supatra; Phusua, Arunee; Suanta, Sudjai; Fanhchaksai, Kanda; Sae-Tung, Rattika; Sanguansermsri, Torpong

2017-12-01

High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR-HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.4 kb beta-globin deletion. We sought to characterize the mutations in carriers and to enable prenatal diagnosis in 126 couples at risk of having a fetus with beta-thalassemia disease. The protocols identified 18 common mutations causing beta-thalassemia, including the rare codon 132 (A-T) mutation. Each mutation showed a specific HRM pattern and all results were in concordance with those from direct DNA sequencing or gap-PCR methods. In cases of beta-thalassemia disease resulting from homozygosity for a mutation or compound heterozygosity for two mutations on the same amplified segment, the HRM patterns were different to those of a single mutation and were specific for each combination. HRM analysis is a simple and useful method for mutation identification in beta-thalassemia carriers and prenatal diagnosis of beta-thalassemia in northern Thailand.

Rapid Identification of Echinococcus granulosus and E. canadensis Using High-Resolution Melting (HRM) Analysis by Focusing on a Single Nucleotide Polymorphism.

PubMed

Safa, Ahmad Hosseini; Harandi, Majid Fasihi; Tajaddini, Mohammadhasan; Rostami-Nejad, Mohammad; Mohtashami-Pour, Mehdi; Pestehchian, Nader

2016-07-22

High-resolution melting (HRM) is a reliable and sensitive scanning method to detect variation in DNA sequences. We used this method to better understand the epidemiology and transmission of Echinococcus granulosus. We tested the use of HRM to discriminate the genotypes of E. granulosus and E. canadensis. One hundred forty-one hydatid cysts were collected from slaughtered animals in different parts of Isfahan-Iran in 2013. After DNA extraction, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using PCR coupled with the HRM curve. The result of HRM analysis using partial the sequences of cox1 gene revealed that 93, 35, and 2 isolates were identified as G1, G3, and G6 genotypes, respectively. A single nucleotide polymorphism (SNP) was found in locus 9867 of the cox1 gene. This is a critical locus for the differentiation between the G6 and G7 genotypes. In the phylogenic tree, the sample with a SNP was located between the G6 and G7 genotypes, which suggest that this isolate has a G6/G7 genotype. The HRM analysis developed in the present study provides a powerful technique for molecular and epidemiological studies on echinococcosis in humans and animals.

Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions

PubMed Central

Wilson, R. Bryce; Davis, Dana; Mitchell, Aaron P.

1999-01-01

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions. PMID:10074081

Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees

PubMed Central

Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

2016-01-01

Background: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Materials and Methods: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. Results: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. Conclusion: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily determine the A. paniculata species in herbal products tested. Abbreviations used: bp: Base pair, Tm: Melting temperature PMID:27041863

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

PubMed

Tulsiani, S M; Craig, S B; Graham, G C; Cobbold, R C; Dohnt, M F; Burns, M-A; Jansen, C C; Leung, L K-P; Field, H E; Smythe, L D

2010-07-01

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Evaluation of esophageal motility utilizing the functional lumen imaging probe (FLIP)

PubMed Central

Carlson, Dustin A.; Kahrilas, Peter J.; Lin, Zhiyue; Hirano, Ikuo; Gonsalves, Nirmala; Listernick, Zoe; Ritter, Katherine; Tye, Michael; Ponds, Fraukje A.; Wong, Ian; Pandolfino, John E.

2016-01-01

Background Esophagogastric junction (EGJ) distensibility and distension-mediated peristalsis can be assessed with the functional lumen imaging probe (FLIP) during a sedated upper endoscopy. We aimed to describe esophageal motility assessment using FLIP topography in patients presenting with dysphagia. Methods 145 patients (ages 18 – 85, 54% female) with dysphagia that completed upper endoscopy with a 16-cm FLIP assembly and high-resolution manometry (HRM) were included. HRM was analyzed according to the Chicago Classification of esophageal motility disorders; major esophageal motility disorders were considered ‘abnormal’. FLIP studies were analyzed using a customized program to calculate the EGJ-distensibility index (DI) and generate FLIP topography plots to identify esophageal contractility patterns. FLIP topography was considered ‘abnormal’ if EGJ-DI was < 2.8 mm2/mmHg or contractility pattern demonstrated absent contractility or repetitive, retrograde contractions. Results HRM was abnormal in 111 (77%) patients: 70 achalasia (19 type I, 39 type II, 12 type III), 38 EGJ outflow obstruction, and three jackhammer esophagus. FLIP topography was abnormal in 106 (95%) of these patients, including all 70 achalasia patients. HRM was ‘normal’ in 34 (23%) patients: five ineffective esophageal motility and 29 normal motility. 17 (50%) had abnormal FLIP topography including 13 (37%) with abnormal EGJ-DI. Conclusions FLIP topography provides a well-tolerated method for esophageal motility assessment (especially to identify achalasia) at the time of upper endoscopy. FLIP topography findings that are discordant with HRM may indicate otherwise undetected abnormalities of esophageal function, thus FLIP provides an alternative and complementary method to HRM for evaluation of non-obstructive dysphagia. PMID:27725650

Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening.

PubMed

Benej, Martin; Bendlova, Bela; Vaclavikova, Eliska; Poturnajova, Martina

2011-10-06

Reliable and effective primary screening of mutation carriers is the key condition for common diagnostic use. The objective of this study is to validate the method high resolution melting (HRM) analysis for routine primary mutation screening and accomplish its optimization, evaluation and validation. Due to their heterozygous nature, germline point mutations of c-RET proto-oncogene, associated to multiple endocrine neoplasia type 2 (MEN2), are suitable for HRM analysis. Early identification of mutation carriers has a major impact on patients' survival due to early onset of medullary thyroid carcinoma (MTC) and resistance to conventional therapy. The authors performed a series of validation assays according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for validation of analytical procedures, along with appropriate design and optimization experiments. After validated evaluation of HRM, the method was utilized for primary screening of 28 pathogenic c-RET mutations distributed among nine exons of c-RET gene. Validation experiments confirm the repeatability, robustness, accuracy and reproducibility of HRM. All c-RET gene pathogenic variants were detected with no occurrence of false-positive/false-negative results. The data provide basic information about design, establishment and validation of HRM for primary screening of genetic variants in order to distinguish heterozygous point mutation carriers among the wild-type sequence carriers. HRM analysis is a powerful and reliable tool for rapid and cost-effective primary screening, e.g., of c-RET gene germline and/or sporadic mutations and can be used as a first line potential diagnostic tool.

Developing Critical Understanding in HRM Students: Using Innovative Teaching Methods to Encourage Deep Approaches to Study

ERIC Educational Resources Information Center

Butler, Michael J. R.; Reddy, Peter

2010-01-01

Purpose: This paper aims to focus on developing critical understanding in human resource management (HRM) students in Aston Business School, UK. The paper reveals that innovative teaching methods encourage deep approaches to study, an indicator of students reaching their own understanding of material and ideas. This improves student employability…

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

PubMed Central

Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia

2013-01-01

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

PubMed

Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

2016-01-01

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

Health resort medicine in non-musculoskeletal disorders: is there evidence of its effectiveness?

NASA Astrophysics Data System (ADS)

Stier-Jarmer, Marita; Kus, Sandra; Frisch, Dieter; Sabariego, Carla; Schuh, Angela

2015-10-01

Health resort medicine (HRM; in German: Kurortmedizin) is a field of medicine with long-lasting tradition in several European countries. A number of systematic reviews have shown the effectiveness of HRM in musculoskeletal conditions. Reviews focusing on the effectiveness of HRM in non-musculoskeletal disorders are rare. This systematic review aims to provide an overview about all types of health resort treatments applied in non-musculoskeletal conditions, to summarize evidence for its effectiveness and to assess the quality of published studies. MEDLINE, Web of Knowledge and Embase were searched for articles published between January 2002 and December 2013. We used a broad search strategy in order to find studies investigating the effects of HRM in non-musculoskeletal disorders. Two authors independently extracted data and assessed quality using the Effective Public Health Practice Project Quality Assessment Tool for Quantitative Studies (EPHPP-QAT). Forty-one studies (19 of them with control group) from eight countries examining the efficacy of various forms of spa treatment for 12 disease groups were included. The studies are markedly heterogeneous regarding study design, population and treatment. HRM treatment is associated with clinical improvement in diseases of the skin, respiratory, circulatory, digestive and nervous system among others. However, small samples, the lack of control groups and an insufficient follow-up often limit the generated evidence. The scientific literature of the last decade has shown that a number of non-musculoskeletal disorders are treated with different kinds of HRM. The challenge for the future will be to carry out thoroughly designed studies in larger patient populations to corroborate the impact of HRM treatment on non-musculoskeletal disorders.

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

PubMed

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

PubMed Central

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S.; Garcia-Closas, Montserrat; Sherman, Mark E.; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P.; Khan, Javed; Chanock, Stephen

2011-01-01

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples. PMID:21264207

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

A novel dNTP-limited PCR and HRM assay to detect Williams-Beuren syndrome.

PubMed

Zhang, Lichen; Zhang, Xiaoqing; You, Guoling; Yu, Yongguo; Fu, Qihua

2018-06-01

Williams-Beuren syndrome (WBS) is caused by a microdeletion of chromosome arm 7q11.23. A rapid and inexpensive genotyping method to detect microdeletion on 7q11.23 needs to be developed for the diagnosis of WBS. This study describes the development of a new type of molecular diagnosis method to detect microdeletion on 7q11.23 based upon high-resolution melting (HRM). Four genes on 7q11.23 were selected as the target genes for the deletion genotyping. dNTP-limited duplex PCR was used to amplify the reference gene, CFTR, and one of the four genes respectively on 7q11.23. An HRM assay was performed on the PCR products, and the height ratio of the negative derivative peaks between the target gene and reference gene was employed to analyze the copy number variation of the target region. A new genotyping method for detecting 7q11.23 deletion was developed based upon dNTP-limited PCR and HRM, which cost only 96 min. Samples from 15 WBS patients and 12 healthy individuals were genotyped by this method in a blinded fashion, and the sensitivity and specificity was 100% (95% CI, 0.80-1, and 95% CI, 0.75-1, respectively) which was proved by CytoScan HD array. The HRM assay we developed is an rapid, inexpensive, and highly accurate method for genotyping 7q11.23 deletion. It is potentially useful in the clinical diagnosis of WBS. Copyright © 2018 Elsevier B.V. All rights reserved.

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

PubMed Central

Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

2011-01-01

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

PubMed

Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

2016-01-01

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data

PubMed Central

Hatae, Ryusuke; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O.; Mizoguchi, Masahiro; Iihara, Koji

2016-01-01

High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

PubMed

Tong, Steven Y C; Xie, Shirley; Richardson, Leisha J; Ballard, Susan A; Dakh, Farshid; Grabsch, Elizabeth A; Grayson, M Lindsay; Howden, Benjamin P; Johnson, Paul D R; Giffard, Philip M

2011-01-01

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Facilitating job retention for chronically ill employees: perspectives of line managers and human resource managers

PubMed Central

2011-01-01

Background Chronic diseases are a leading contributor to work disability and job loss in Europe. Recent EU policies aim to improve job retention among chronically ill employees. Disability and occupational health researchers argue that this requires a coordinated and pro-active approach at the workplace by occupational health professionals, line managers (LMs) and human resource managers (HRM). Little is known about the perspectives of LMs an HRM on what is needed to facilitate job retention among chronically ill employees. The aim of this qualitative study was to explore and compare the perspectives of Dutch LMs and HRM on this issue. Methods Concept mapping methodology was used to elicit and map statements (ideas) from 10 LMs and 17 HRM about what is needed to ensure continued employment for chronically ill employees. Study participants were recruited through a higher education and an occupational health services organization. Results Participants generated 35 statements. Each group (LMs and HRM) sorted these statements into six thematic clusters. LMs and HRM identified four similar clusters: LMs and HRM must be knowledgeable about the impact of chronic disease on the employee; employees must accept responsibility for work retention; work adaptations must be implemented; and clear company policy. Thematic clusters identified only by LMs were: good manager/employee cooperation and knowledge transfer within the company. Unique clusters identified by HRM were: company culture and organizational support. Conclusions There were both similarities and differences between the views of LMs and HRM on what may facilitate job retention for chronically ill employees. LMs perceived manager/employee cooperation as the most important mechanism for enabling continued employment for these employees. HRM perceived organizational policy and culture as the most important mechanism. The findings provide information about topics that occupational health researchers and planners should address in developing job retention programs for chronically ill workers. PMID:21586139

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Evaluation of a DNA-based method for spice/herb authentication, so you do not have to worry about what is in your curry, buon appetito!

PubMed

Osathanunkul, Maslin; Ounjai, Sarawut; Osathanunkul, Rossarin; Madesis, Panagiotis

2017-01-01

It is long believed that some spices may help protect against certain chronic conditions. Spices are usually parts of plants that have been powdered into small pieces. Have you ever wondered what the curry powder in your dish is made of? The aim of this work was to develop an appropriate DNA-based method for assessment of spice identity. Selecting the best marker for species recognition in the Zingiberaceae family. Six DNA regions were investigated in silico, including ITS, matK, rbcL, rpoC, trnH-psbA and trnL. Then, only four regions (ITS, matK, rbcL and trnH-psbA) were included in the simulated HRM (High-resolution Melting) analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae species in HRM analysis based on both the percentage of nucleotide variation and GC content. Simulated HRM analysis was performed to test the feasibility of Bar-HRM. We found that ITS2 is the most effective region to be used for identification of the studied species and thus was used in laboratory HRM analysis. All seven tested Zingiberaceae plants were then able to be distinguished using the ITS2 primers in laboratory HRM. Most importantly the melting curves gained from fresh and dried tissue overlapped, which is a crucial outcome for the applicability of the analysis. The method could be used in an authentication test for dried products. In the authentication test, only one of seven store-sold Zingiberaceae products that were tested contained the species listed on their labels, while we found substitution/contamination of the tested purchased products in the rest.

Evaluation of a DNA-based method for spice/herb authentication, so you do not have to worry about what is in your curry, buon appetito!

PubMed Central

Ounjai, Sarawut; Osathanunkul, Rossarin; Madesis, Panagiotis

2017-01-01

It is long believed that some spices may help protect against certain chronic conditions. Spices are usually parts of plants that have been powdered into small pieces. Have you ever wondered what the curry powder in your dish is made of? The aim of this work was to develop an appropriate DNA-based method for assessment of spice identity. Selecting the best marker for species recognition in the Zingiberaceae family. Six DNA regions were investigated in silico, including ITS, matK, rbcL, rpoC, trnH-psbA and trnL. Then, only four regions (ITS, matK, rbcL and trnH-psbA) were included in the simulated HRM (High-resolution Melting) analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae species in HRM analysis based on both the percentage of nucleotide variation and GC content. Simulated HRM analysis was performed to test the feasibility of Bar-HRM. We found that ITS2 is the most effective region to be used for identification of the studied species and thus was used in laboratory HRM analysis. All seven tested Zingiberaceae plants were then able to be distinguished using the ITS2 primers in laboratory HRM. Most importantly the melting curves gained from fresh and dried tissue overlapped, which is a crucial outcome for the applicability of the analysis. The method could be used in an authentication test for dried products. In the authentication test, only one of seven store-sold Zingiberaceae products that were tested contained the species listed on their labels, while we found substitution/contamination of the tested purchased products in the rest. PMID:29020084

High-resolution metabolomics assessment of military personnel: Evaluating analytical strategies for chemical detection

PubMed Central

Liu, Ken H.; Walker, Douglas I.; Uppal, Karan; Tran, ViLinh; Rohrbeck, Patricia; Mallon, Timothy M.; Jones, Dean P.

2016-01-01

Objective To maximize detection of serum metabolites with high-resolution metabolomics (HRM). Methods Department of Defense Serum Repository (DoDSR) samples were analyzed using ultra-high resolution mass spectrometry with three complementary chromatographic phases and four ionization modes. Chemical coverage was evaluated by number of ions detected and accurate mass matches to a human metabolomics database. Results Individual HRM platforms provided accurate mass matches for up to 58% of the KEGG metabolite database. Combining two analytical methods increased matches to 72%, and included metabolites in most major human metabolic pathways and chemical classes. Detection and feature quality varied by analytical configuration. Conclusions Dual chromatography HRM with positive and negative electrospray ionization provides an effective generalized method for metabolic assessment of military personnel. PMID:27501105

[Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

PubMed

Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

2017-08-10

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. 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. 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. 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.

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis.

PubMed

van Blerk, G N; Leibach, L; Mabunda, A; Chapman, A; Louw, D

2011-01-01

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

PubMed

Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

2017-09-19

Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy.

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

PubMed

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz; Strapagiel, Dominik

2017-11-03

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

PubMed

Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

2017-11-22

Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

PubMed Central

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz

2017-01-01

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup. PMID:29099791

Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.

PubMed

Chatzidimopoulos, Michael; Ganopoulos, Ioannis; Vellios, Evangelos; Madesis, Panagiotis; Tsaftaris, Athanasios; Pappas, Athanassios C

2014-11-01

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

PubMed Central

Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

2012-01-01

Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538

High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples.

PubMed

Morinha, Francisco; Travassos, Paulo; Seixas, Fernanda; Santos, Nuno; Sargo, Roberto; Sousa, Luís; Magalhães, Paula; Cabral, João A; Bastos, Estela

2013-05-01

High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing. © 2013 Blackwell Publishing Ltd.

Evaluation of Esophageal Motility Utilizing the Functional Lumen Imaging Probe.

PubMed

Carlson, Dustin A; Kahrilas, Peter J; Lin, Zhiyue; Hirano, Ikuo; Gonsalves, Nirmala; Listernick, Zoe; Ritter, Katherine; Tye, Michael; Ponds, Fraukje A; Wong, Ian; Pandolfino, John E

2016-12-01

Esophagogastric junction (EGJ) distensibility and distension-mediated peristalsis can be assessed with the functional lumen imaging probe (FLIP) during a sedated upper endoscopy. We aimed to describe esophageal motility assessment using FLIP topography in patients presenting with dysphagia. In all, 145 patients (aged 18-85 years, 54% female) with dysphagia that completed upper endoscopy with a 16-cm FLIP assembly and high-resolution manometry (HRM) were included. HRM was analyzed according to the Chicago Classification of esophageal motility disorders; major esophageal motility disorders were considered "abnormal". FLIP studies were analyzed using a customized program to calculate the EGJ-distensibility index (DI) and generate FLIP topography plots to identify esophageal contractility patterns. FLIP topography was considered "abnormal" if EGJ-DI was <2.8 mm 2 /mm Hg or contractility pattern demonstrated absent contractility or repetitive, retrograde contractions. HRM was abnormal in 111 (77%) patients: 70 achalasia (19 type I, 39 type II, and 12 type III), 38 EGJ outflow obstruction, and three jackhammer esophagus. FLIP topography was abnormal in 106 (95%) of these patients, including all 70 achalasia patients. HRM was "normal" in 34 (23%) patients: five ineffective esophageal motility and 29 normal motility. In all, 17 (50%) had abnormal FLIP topography including 13 (37%) with abnormal EGJ-DI. FLIP topography provides a well-tolerated method for esophageal motility assessment (especially to identify achalasia) at the time of upper endoscopy. FLIP topography findings that are discordant with HRM may indicate otherwise undetected abnormalities of esophageal function, thus FLIP provides an alternative and complementary method to HRM for evaluation of non-obstructive dysphagia.

High Resolution Melt analysis for mutation screening in PKD1 and PKD2

PubMed Central

2011-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes. PMID:22008521

Comparison of the college alumnus questionnaire physical activity index with objective monitoring.

PubMed

Strath, Scott J; Bassett, David R; Swartz, Ann M

2004-07-01

Two methods of measuring physical activity (PA) were compared over a consecutive 7-day period among 25 adults (12 men and 13 women). Each day estimates of energy expended in light, moderate, vigorous, and total PA were derived from the simultaneous heart-rate motion sensor (HR+M) technique. At the end of the 7-day period participants completed the College Alumnus Questionnaire Physical Activity Index (CAQ-PAI) and results were compared with HR+M technique estimates. Correlations between the two methods in the four activity categories ranged from r=0.20 to r=0.47, with vigorous and total PA showing higher associations than light and moderate PA. Mean levels of PA (MET-minxwk(-1)) obtained using the two methods were similar in the moderate and vigorous categories, but individual differences were large. Energy expended in light PA was significantly underestimated on the CAQ-PAI, resulting in lower total activity scores on this questionnaire as compared with the HR+M. The CAQ-PAI accurately reflected mean moderate and vigorous activity in comparison with the HR+M technique. The results are consistent with other studies which have shown that physical activity questionnaires are better at assessing vigorous PA than ubiquitous light-moderate activities.

Applying high-resolution melting (HRM) technology to olive oil and wine authenticity.

PubMed

Pereira, Leonor; Gomes, Sónia; Barrias, Sara; Fernandes, José Ramiro; Martins-Lopes, Paula

2018-01-01

Olive oil and wine production have a worldwide economic impact. Their market reliability is under great concern because of the increasing number of fraud and adulteration attempts. The need for a traceability system in all its extension is crucial particularly for the cases of olive oils and wines with certified labels, in which only a limited number of olives and grapevine varieties, respectively, are allowed in a restricted well-defined geographical area. Molecular markers have been vastly applied to the food sector, and in particular High-Resolution DNA Melting technology has been successfully applied for olive oil and wine authentication, as part of the traceability system. In this review, the applications of HRM and their usefulness for this sector considering, Safety, Security and Authenticity will be reviewed. A broad overview of the HRM technique will be presented, focusing on the aspects that are crucial for its success, in particular the new generation of fluorescent dsDNA dyes used for amplicon detection and quantification, and the data analysis. A brief outlook on the olive oil and wine authenticity procedures, based on new DNA technology advances, and in which way this may influence the future establishment of a traceability system will be discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Identification of a HPGD mutation in three families affected with primary hypertrophic osteoarthropathy].

PubMed

Zhang, Wanying; Wang, Tao; Huang, Shuaiwu; Zhao, Xiuli

2018-04-10

To detect mutation of HPGD gene among three pedigrees affected with primary hypertrophic osteoarthropathy (PHO) by DNA sequencing and high-resolution melting (HRM) analysis. Genomic DNA was extracted from peripheral blood samples collected from the pedigrees. PCR and direct sequencing were carried out to identify potential mutations of the HPGD gene. Amplicons containing the mutation spot were generated by nested PCR. The products were then subjected to HRM analysis using the HR-1 instrument. Direct sequencing was carried out in family members and healthy individuals to confirm the result of HRM analysis. A homozygous mutation c.310_311delCT was detected in 2 affected probands, while a heterozygous mutation c.310_311delCT was detected in the third proband. HRM analysis of the fragments encompassing HPGD exon 3 showed 3 curve patterns representing three different genotypes, i.e., the wild type, the c.310_311delCT homozygote, and the c.310_311delCT heterozygote. Result of DNA sequencing was consistent with that of the HRM analysis and phenotype of the subjects. The c.310_311delCT mutation may be the most prevalent mutation among Chinese population. HRM analysis has provided an optimized method for genetic testing of HPGD mutation for its simplicity, rapid turnover and high sensitivity.

Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.).

PubMed

Kamel, Katarzyna A; Kroc, Magdalena; Święcicki, Wojciech

2015-01-01

Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lupinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.

Bar-HRM for Authentication of Plant-Based Medicines: Evaluation of Three Medicinal Products Derived from Acanthaceae Species

PubMed Central

Osathanunkul, Maslin; Madesis, Panagiotis; de Boer, Hugo

2015-01-01

Medicinal plants are used as a popular alternative to synthetic drugs, both in developed and developing countries. The economic importance of the herbal and natural supplement industry is increasing every year. As the herbal industry grows, consumer safety is one issue that cannot be overlooked. Herbal products in Thai local markets are commonly sold without packaging or labels. Plant powders are stored in large bags or boxes, and therefore buying local herbal products poses a high risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate three medicinal Acanthaceae species (Acanthus ebracteatus, Andrographis paniculata and Rhinacanthus nasutus) commonly used in Thailand. The rbcL barcode was selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Melting data from the HRM assay using the designed rbcL primers showed that the three chosen species could be distinguished from each other. HRM curves of all fifteen test samples indicated that three of tested products did not contain the indicated species. Two closely related species (A. paniculata and R. nasutus), which have a high level of morphological similarity, were interchanged with one another in three tested products. Incorrect information on packaging and labels of the tested herbal products was the cause of the results shown here. Morphological similarity among the species of interest also hindered the collection process. The Bar-HRM method developed here proved useful in aiding in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products. PMID:26011474

Bar-HRM for Authentication of Plant-Based Medicines: Evaluation of Three Medicinal Products Derived from Acanthaceae Species.

PubMed

Osathanunkul, Maslin; Madesis, Panagiotis; de Boer, Hugo

2015-01-01

Medicinal plants are used as a popular alternative to synthetic drugs, both in developed and developing countries. The economic importance of the herbal and natural supplement industry is increasing every year. As the herbal industry grows, consumer safety is one issue that cannot be overlooked. Herbal products in Thai local markets are commonly sold without packaging or labels. Plant powders are stored in large bags or boxes, and therefore buying local herbal products poses a high risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate three medicinal Acanthaceae species (Acanthus ebracteatus, Andrographis paniculata and Rhinacanthus nasutus) commonly used in Thailand. The rbcL barcode was selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Melting data from the HRM assay using the designed rbcL primers showed that the three chosen species could be distinguished from each other. HRM curves of all fifteen test samples indicated that three of tested products did not contain the indicated species. Two closely related species (A. paniculata and R. nasutus), which have a high level of morphological similarity, were interchanged with one another in three tested products. Incorrect information on packaging and labels of the tested herbal products was the cause of the results shown here. Morphological similarity among the species of interest also hindered the collection process. The Bar-HRM method developed here proved useful in aiding in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.

Use of Case Study Methods in Human Resource Management, Development, and Training Courses: Strategies and Techniques

ERIC Educational Resources Information Center

Maxwell, James R.; Gilberti, Anthony F.; Mupinga, Davison M.

2006-01-01

This paper will study some of the problems associated with case studies and make recommendations using standard and innovative methodologies effectively. Human resource management (HRM) and resource development cases provide context for analysis and decision-making designs in different industries. In most HRM development and training courses…

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

PubMed

Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

2012-09-01

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

PubMed Central

Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob; Feld, Louise; Holben, William E.

2014-01-01

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition. PMID:24610853

Differentiation of five enterohepatic Helicobacter species by nested PCR with high-resolution melting curve analysis.

PubMed

Wu, Miaoli; Rao, Dan; Zhu, Yujun; Wang, Jing; Yuan, Wen; Zhang, Yu; Huang, Ren; Guo, Pengju

2017-04-01

Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species. © 2016 John Wiley & Sons Ltd.

Rapid identification of apolipoprotein E genotypes by high-resolution melting analysis in Chinese Han and African Fang populations.

PubMed

Zhan, Xiu-Hui; Zha, Guang-Cai; Jiao, Ji-Wei; Yang, Li-Ye; Zhan, Xiao-Fen; Chen, Jiang-Tao; Xie, Dong-DE; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Obono, Maximo Miko Ondo; Ehapo, Carlos Sala; Wei, Er-Jia; Zheng, Yu-Zhong; Yang, Hui; Lin, Min

2015-02-01

Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P<0.05). In conclusion, the present study revealed the molecular characterization of APOE gene polymorphism in the Han population from the Chaozhou region of Southern China and the Fang population from Equatorial Guinea. The findings of the study indicated that HRM analysis could be used as an accurate and sensitive method for the rapid screening and identification of APOE genotypes in prospective clinical and population genetic analyses.

Managing hospital doctors and their practice: what can we learn about human resource management from non-healthcare organisations?

PubMed

Trebble, Timothy M; Heyworth, Nicola; Clarke, Nicholas; Powell, Timothy; Hockey, Peter M

2014-11-21

Improved management of clinicians' time and practice is advocated to address increasing demands on healthcare provision in the UK National Health Service (NHS). Human resource management (HRM) is associated with improvements in organisational performance and outcomes within and outside of healthcare, but with limited use in managing individual clinicians. This may reflect the absence of effective and transferrable models. The current systems of managing the performance of individual clinicians in a secondary healthcare organisation were reviewed through the study of practice in 10 successful partnership organisations, including knowledge worker predominant, within commercial, public and voluntary sector operating environments. Reciprocal visits to the secondary healthcare environment were undertaken. Six themes in performance related HRM were identified across the external organisations representing best practice and considered transferrable to managing clinicians in secondary care organisations. These included: performance measurement through defined outcomes at the team level with decision making through local data interpretation; performance improvement through empowered formal leadership with organisational support; individual performance review (IPR); and reward, recognition and talent management. The role of the executive was considered essential to support and implement effective HRM, with management of staff performance, behaviour and development integrated into organisational strategy, including through the use of universally applied values and effective communication. These approaches reflected many of the key aspects of high performance work systems and strategic HRM. There is the potential to develop systems of HRM of individual clinicians in secondary healthcare to improve practice. This should include both performance measurement and performance improvement but also engagement at an organisational level. This suggests that effective HRM and performance management of individual clinicians may be possible but requires an alternative approach for the NHS.

High-Resolution Metabolomics Assessment of Military Personnel: Evaluating Analytical Strategies for Chemical Detection.

PubMed

Liu, Ken H; Walker, Douglas I; Uppal, Karan; Tran, ViLinh; Rohrbeck, Patricia; Mallon, Timothy M; Jones, Dean P

2016-08-01

The aim of this study was to maximize detection of serum metabolites with high-resolution metabolomics (HRM). Department of Defense Serum Repository (DoDSR) samples were analyzed using ultrahigh resolution mass spectrometry with three complementary chromatographic phases and four ionization modes. Chemical coverage was evaluated by number of ions detected and accurate mass matches to a human metabolomics database. Individual HRM platforms provided accurate mass matches for up to 58% of the KEGG metabolite database. Combining two analytical methods increased matches to 72% and included metabolites in most major human metabolic pathways and chemical classes. Detection and feature quality varied by analytical configuration. Dual chromatography HRM with positive and negative electrospray ionization provides an effective generalized method for metabolic assessment of military personnel.

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis.

PubMed

Haeili, M; Fooladi, A I; Bostanabad, S Z; Sarokhalil, D D; Siavoshi, F; Feizabadi, M M

2014-01-01

Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.

High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR.

PubMed

Iacumin, Lucilla; Ginaldi, Federica; Manzano, Marisa; Anastasi, Veronica; Reale, Anna; Zotta, Teresa; Rossi, Franca; Coppola, Raffaele; Comi, Giuseppe

2015-04-01

The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetic high throughput screening in Retinitis Pigmentosa based on high resolution melting (HRM) analysis.

PubMed

Anasagasti, Ander; Barandika, Olatz; Irigoyen, Cristina; Benitez, Bruno A; Cooper, Breanna; Cruchaga, Carlos; López de Munain, Adolfo; Ruiz-Ederra, Javier

2013-11-01

Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n = 96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4% of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for medium sized (<4 kb transcript) RP genes, which constitute over 80% of the total of known RP genes.

Genetic highthroughput screening in retinitis pigmentosa based on high resolution melting (HRM) analysis.

PubMed

Anasagasti, Ander; Barandika, Olatz; Irigoyen, Cristina; Benitez, Bruno A; Cooper, Breanna; Cruchaga, Carlos; López de Munain, Adolfo; Ruiz-Ederra, Javier

2013-10-24

Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n=96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4 % of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for medium sized (<4 kb transcript) RP genes, which constitute over 80% of the total of known RP genes. © 2013 Published by Elsevier Ltd.

The Effects of Bolus Volume and Texture on Pharyngeal Pressure Events Using High-resolution Manometry and Its Comparison with Videofluoroscopic Swallowing Study

PubMed Central

Ryu, Ju Seok; Park, Donghwi; Oh, Yoongul; Lee, Seok Tae; Kang, Jin Young

2016-01-01

Background/Aims The purpose of this study was to develop new parameters of high-resolution manometry (HRM) and to applicate these to quantify the effect of bolus volume and texture on pharyngeal swallowing. Methods Ten healthy subjects prospectively swallowed dry, thin fluid 2 mL, thin fluid 5 mL, thin fluid 10 mL, and drinking twice to compare effects of bolus volume. To compare effect of texture, subjects swallowed thin fluid 5 mL, yogurt 5 mL, and bread twice. A 32-sensor HRM catheter and BioVIEW ANALYSIS software were used for data collection and analysis. HRM data were synchronized with kinematic analysis of videofluoroscopic swallowing study (VFSS) using epiglottis tilting. Results Linear correlation analysis for volume showed significant correlation for area of velopharynx, duration of velopharynx, pre-upper esophageal sphincter (UES) maximal pressure, minimal UES pressure, UES activity time, and nadir UES duration. In the correlation with texture, all parameters were not significantly different. The contraction of the velopharynx was faster than laryngeal elevation. The durations of UES relaxation was shorter in the kinematic analysis than HRM. Conclusions The bolus volume was shown to have significant effect on pharyngeal pressure and timing, but the texture did not show any effect on pharyngeal swallowing. The parameters of HRM were more sensitive than those of kinematic analysis. As the parameters of HRM are based on precise anatomic structure and the kinematic analysis reflects the actions of multiple anatomic structures, HRM and VFSS should be used according to their purposes. PMID:26598598

Measurement of Dynamic Urethral Pressures with a High Resolution Manometry System in Continent and Incontinent Women

PubMed Central

Kirby, Anna C; Tan-Kim, Jasmine; Nager, Charles W.

2015-01-01

Objectives Female stress urinary incontinence (SUI) is caused by urethral dysfunction during dynamic conditions, but current technology has limitations in measuring urethral pressures under dynamic conditions. An 8-French high resolution manometry catheter (HRM) currently in clinical use in gastroenterology may accurately measure urethral pressures under dynamic conditions because it has a 25ms response rate and circumferential pressure sensors along the length of the catheter (ManoScan® ESO, Given Imaging). We evaluated the concordance, repeatability, and tolerability of this catheter. Methods We measured resting, cough, and strain maximum urethral closure pressures (MUCPs) using HRM and measured resting MUCPs with water perfusion side-hole catheter urethral pressure profilometry (UPP) in 37 continent and 28 stress incontinent subjects. Maneuvers were repeated after moving the HRM catheter along the urethral length to evaluate whether results depend on catheter positioning. Visual analog pain scores evaluated the comfort of HRM compared to UPP. Results The correlation coefficient for resting MUCPs measured by HRM vs. UPP was high (r = 0.79, p<0.001). Repeatability after catheter repositioning was high for rest, cough, and strain with HRM: r= 0.92, 0.89, and 0.89. Mean MUCPs (rest, cough, strain) were higher in continent than incontinent subjects (all p < 0.001) and decreased more in incontinent subjects than continent subjects during cough and strain maneuvers compared to rest. Conclusions This preliminary study shows that HRM is concordant with standard technology, repeatable, and well tolerated in the urethra. Incontinent women have more impairment of their urethral closure pressures during cough and strain than continent women. PMID:25185595

Understanding human resource management practices in Botswana's public health sector.

PubMed

Seitio-Kgokgwe, Onalenna Stannie; Gauld, Robin; Hill, Philip C; Barnett, Pauline

2016-11-21

Purpose The purpose of this paper is to assess the management of the public sector health workforce in Botswana. Using institutional frameworks it aims to document and analyse human resource management (HRM) practices, and make recommendations to improve employee and health system outcomes. Design/methodology/approach The paper draws from a large study that used a mixed methods approach to assess performance of Botswana's Ministry of Health (MOH). It uses data collected through document analysis and in-depth interviews of 54 key informants comprising policy makers, senior staff of the MOH and its stakeholder organizations. Findings Public health sector HRM in Botswana has experienced inadequate planning, poor deployment and underutilization of staff. Lack of comprehensive retention strategies and poor working conditions contributed to the failure to attract and retain skilled personnel. Relationships with both formal and informal environments affected HRM performance. Research limitations/implications While document review was a major source of data for this paper, the weaknesses in the human resource information system limited availability of data. Practical implications This paper presents an argument for the need for consideration of formal and informal environments in developing effective HRM strategies. Originality/value This research provides a rare system-wide approach to health HRM in a Sub-Saharan African country. It contributes to the literature and evidence needed to guide HRM policy decisions and practices.

ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger.

PubMed

Xiong, Chao; Hu, Zhi-Gang; Tu, Yuan; Liu, He-Gang; Wang, Ping; Zhao, Ming-Ming; SHIi, Yu-Hua; Wu, Lan; Sun, Wei; Chen, Shi-Lin

2016-12-01

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. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.

PubMed

Carbonell, Pablo; Turpin, María C; Torres-Moreno, Daniel; Molina-Martínez, Irene; García-Solano, José; Perez-Guillermo, Miguel; Conesa-Zamora, Pablo

2011-09-01

The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffin-embedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent (κ = 0.9411 and κ = 0.8988, respectively); for HRM, the concordances were good (κ = 0.7973 and κ = 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in archival paraffin-embedded tissues. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

PubMed Central

Anthwal, Divya; Gupta, Rakesh Kumar; Bhalla, Manpreet; Bhatnagar, Shinjini

2017-01-01

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INH-inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases. PMID:28330890

High-Resolution Manometry Improves the Diagnosis of Esophageal Motility Disorders in Patients With Dysphagia: A Randomized Multicenter Study.

PubMed

Roman, Sabine; Huot, Laure; Zerbib, Frank; Bruley des Varannes, Stanislas; Gourcerol, Guillaume; Coffin, Benoit; Ropert, Alain; Roux, Adeline; Mion, François

2016-03-01

High-resolution manometry (HRM) might be superior to conventional manometry (CM) to diagnose esophageal motility disorders. We aimed to compare the diagnosis performed with HRM and CM and confirmed at 6 months in a multicenter randomized trial. Patients with unexplained dysphagia were randomized to undergo either CM or HRM. Motility disorders were diagnosed using the Castell and Spechler classification for CM and the Chicago classification for HRM. Diagnosis confirmation was based on clinical outcome and response to treatment after 6-month follow-up. The initial diagnosis and percentage of confirmed diagnoses were compared between the two arms (CM and HRM). In total, 247 patients were randomized and 245 analyzed: 122 in the CM arm and 123 in the HRM arm. A manometric diagnosis was more frequently initially achieved with HRM than with CM (97% vs. 84%; P<0.01). Achalasia was more frequent in the HRM arm (26% vs. 12% in the CM arm; P<0.01) while normal examinations were more frequent in the CM arm (52% vs. 28% in the HRM arm; P<0.05). After follow-up, the initial diagnosis was confirmed in 89% of patients in the HRM arm vs. 81% in the CM arm (P=0.07). Finally, overall procedure tolerance was better with CM than with HRM (P<0.01). This randomized trial demonstrated an improved diagnostic yield for achalasia with HRM compared with CM. Diagnoses tended to be more frequently confirmed in patients who underwent HRM, suggesting that esophageal motility disorders could be identified earlier with HRM than with CM (ClinicalTrial.gov, NCT01284894).

Quantitative Analysis of Swallowing Function Between Dysphagia Patients and Healthy Subjects Using High-Resolution Manometry

PubMed Central

2017-01-01

Objective To compare swallowing function between healthy subjects and patients with pharyngeal dysphagia using high resolution manometry (HRM) and to evaluate the usefulness of HRM for detecting pharyngeal dysphagia. Methods Seventy-five patients with dysphagia and 28 healthy subjects were included in this study. Diagnosis of dysphagia was confirmed by a videofluoroscopy. HRM was performed to measure pressure and timing information at the velopharynx (VP), tongue base (TB), and upper esophageal sphincter (UES). HRM parameters were compared between dysphagia and healthy groups. Optimal threshold values of significant HRM parameters for dysphagia were determined. Results VP maximal pressure, TB maximal pressure, UES relaxation duration, and UES resting pressure were lower in the dysphagia group than those in healthy group. UES minimal pressure was higher in dysphagia group than in the healthy group. Receiver operating characteristic (ROC) analyses were conducted to validate optimal threshold values for significant HRM parameters to identify patients with pharyngeal dysphagia. With maximal VP pressure at a threshold value of 144.0 mmHg, dysphagia was identified with 96.4% sensitivity and 74.7% specificity. With maximal TB pressure at a threshold value of 158.0 mmHg, dysphagia was identified with 96.4% sensitivity and 77.3% specificity. At a threshold value of 2.0 mmHg for UES minimal pressure, dysphagia was diagnosed at 74.7% sensitivity and 60.7% specificity. Lastly, UES relaxation duration of <0.58 seconds had 85.7% sensitivity and 65.3% specificity, and UES resting pressure of <75.0 mmHg had 89.3% sensitivity and 90.7% specificity for identifying dysphagia. Conclusion We present evidence that HRM could be a useful evaluation tool for detecting pharyngeal dysphagia. PMID:29201816

Human resource management and performance in healthcare organisations.

PubMed

Harris, Claire; Cortvriend, Penny; Hyde, Paula

2007-01-01

The purpose of this paper is to compare the evidence from a range of reviews concerned with the links between human resource management (HRM) and performance. The aim of the paper is to review this diverse literature, and to derive human resource (HR) implications for healthcare researchers, policy makers and managers. Recent reviews of the human resource management and performance literature are examined, in addition to the inclusion of a previously unpublished review. Their methods, HRM focus, findings and recommendations are contrasted in order to produce this review. The paper finds that relationships have been found between a range of HRM practices, policies systems and performance. Despite being an important concern for HR professionals, there is little research exploring the link between HRM and performance in the health sector. The paper sees that recent studies have found HRM practices to be associated with patient outcomes such as mortality, yet they yield little information regarding the processes through which HRM affects individual performance and its consequent impact on patient care. The use of approaches that seek to gain an understanding of workers' interpretations of their experience, i.e. the psychological process through which HRM can affect individual performance, may shed some light on how these processes work in practice. The paper shows that increasing autonomy for healthcare organisations in the UK, i.e. Foundation Trusts, may offer increased opportunity for locally tailored HR systems and practices. The paper presents findings drawn from a review of previous research on a subject of increasing relevance to HR researchers and practitioners in healthcare organisations. The paper indicates alternative approaches to research and practice in light of extant research.

The Effect of Blending HRM Transformational Leadership Style with HRM ICT Expertise Leadership Style on Creating New HRM Strategy That Enable National Companies to Go Global. Evidence from Jordan: Sayegh Group and Hikma Pharmaceutical Corporation

ERIC Educational Resources Information Center

Khudeir, Hamzeh

2016-01-01

There is no single HRM strategy that can cure all HRM problems and complicated issues. Thus, most companies go by the book in this regard; however, the majorities have their own policies and strategies they use to achieve their objectives in general and competitive advantage in particular. One of the strategies used in HRM is blending HRM…

Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene.

PubMed

Lopez-Oceja, A; Nuñez, C; Baeta, M; Gamarra, D; de Pancorbo, M M

2017-12-15

Meat adulteration by substitution with lower value products and/or mislabeling involves economic, health, quality and socio-religious issues. Therefore, identification and traceability of meat species has become an important subject to detect possible fraudulent practices. In the present study the development of a high resolution melt (HRM) screening method for the identification of eight common meat species is reported. Samples from Bos taurus, Ovis aries, Sus scrofa domestica, Equus caballus, Oryctolagus cuniculus, Gallus gallus domesticus, Meleagris gallopavo and Coturnix coturnix were analyzed through the amplification of a 148 bp fragment from the cyt b gene with a universal primer pair in HRM analyses. Melting profiles from each species, as well as from several DNA mixtures of these species and blind samples, allowed a successful species differentiation. The results demonstrated that the HRM method here proposed is a fast, reliable, and low-cost screening technique. Copyright © 2017 Elsevier Ltd. All rights reserved.

High resolution melting analysis: rapid and precise characterisation of recombinant influenza A genomes

PubMed Central

2013-01-01

Background High resolution melting analysis (HRM) is a rapid and cost-effective technique for the characterisation of PCR amplicons. Because the reverse genetics of segmented influenza A viruses allows the generation of numerous influenza A virus reassortants within a short time, methods for the rapid selection of the correct recombinants are very useful. Methods PCR primer pairs covering the single nucleotide polymorphism (SNP) positions of two different influenza A H5N1 strains were designed. Reassortants of the two different H5N1 isolates were used as a model to prove the suitability of HRM for the selection of the correct recombinants. Furthermore, two different cycler instruments were compared. Results Both cycler instruments generated comparable average melting peaks, which allowed the easy identification and selection of the correct cloned segments or reassorted viruses. Conclusions HRM is a highly suitable method for the rapid and precise characterisation of cloned influenza A genomes. PMID:24028349

Radiofrequency Catheter Ablation for Atrial Fibrillation Elicited "Jackhammer Esophagus": A New Complication Due to Vagal Nerve Stimulation?

PubMed

Tolone, Salvatore; Savarino, Edoardo; Docimo, Ludovico

2015-10-01

Radiofrequency catheter ablation (RFCA) is a potentially curative method for treatment of highly symptomatic and drug-refractory atrial fibrillation (AF). However, this technique can provoke esophageal and nerve lesion, due to thermal injury. To our knowledge, there have been no reported cases of a newly described motor disorder, the Jackhammer esophagus (JE) after RFCA, independently of GERD. We report a case of JE diagnosed by high-resolution manometry (HRM), in whom esophageal symptoms developed 2 weeks after RFCA, in absence of objective evidence of GERD. A 65-year-old male with highly symptomatic, drug-refractory paroxysmal AF was candidate to complete electrical pulmonary vein isolation with RFCA. Prior the procedure, the patient underwent HRM and impedance-pH to rule out GERD or hiatal hernia presence. All HRM parameters, according to Chicago classification, were within normal limits. No significant gastroesophageal reflux was documented at impedance pH monitoring. Patient underwent RFCA with electrical disconnection of pulmonary vein. After two weeks, patient started to complain of dysphagia for solids, with acute chest-pain. The patient repeated HRM and impedance-pH monitoring 8 weeks after RFCA. HRM showed in all liquid swallows the typical spastic hypercontractile contractions consistent with the diagnosis of JE, whereas impedance-pH monitoring resulted again negative for GERD. Esophageal dysmotility can represent a possible complication of RFCA for AF, probably due to a vagal nerve injury, and dysphagia appearance after this procedure must be timely investigated by HRM.

Right place of human resource management in the reform of health sector.

PubMed

Hassani, Seyed Abas; Mobaraki, Hossein; Bayat, Maboubeh; Mafimoradi, Shiva

2013-01-01

In this paper the real role and place of human resource (HR) in health system reform will be discussed and determined within the whole system through the comprehensive Human Resource Management (HRM) model. Delphi survey and a questionnaire were used to 1) collect HR manager ideas and comments and 2) identify the main challenges of HRM. Then the results were discussed in an expert panel after being analyzed by content analysis method. Also, a deep focus study of recorded documents related to Health Human Resource Management was done. Then based on all achieved results, a rich picture was drawn to illustrate the right place of HRM in health sector. Finally, the authors revitalize the missed function of HRM within the health sector by drawing a holistic conceptual model. The most percentage of frequency about HR belongs to "Lack of reliable HR information system" (91%) and the least percentage of frequency belongs to "Low responsibility of HR" (28%). The most percentage of frequency about HR manager belongs to "Inattention to HR managers as key managers and consider them in background" (80%) and the least percentage of frequency belongs to "Lack of coordination between universities' policies" (30%). According to the conceptual framework, human resources employed in health system are viewed from two comprehensive approaches: instrumental approach and institutional. Unlike the common belief that looks HRM through the supportive approach, it is discussed that HRM not only has an instrumental role, but also do have a driver role.

High resolution melting curve assay for detecting rs12979860 IL28B polymorphisms involved in response of Iranian patients to chronic hepatitis C treatment.

PubMed

Fateh, Abolfazl; Aghasadeghi, Mohammad Reza; Keyvani, Hossein; Mollaie, Hamid Reza; Yari, Shamsi; Hadizade Tasbiti, Ali Reza; Ghazanfari, Morteza; Monavari, Seyed Hamid Reza

2015-01-01

A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful.

Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

PubMed

Xiao, Zhujun; Li, Bingsheng; Wang, Guozhen; Zhu, Weisi; Wang, Zhongqiu; Lin, Jinfeng; Xu, Angao; Wang, Xinying

2014-04-20

Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (P<0.001, R(2)=0.957) and VIM (P<0.001, R(2)=0.954). MS-HRM shows potential for CRC screening. Copyright © 2014 Elsevier B.V. All rights reserved.

High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

PubMed

Dos Santos Rocha, Alípio; de Amorim, Isis Salviano Soares; Simão, Tatiana de Almeida; da Fonseca, Adenilson de Souza; Garrido, Rodrigo Grazinoli; Mencalha, Andre Luiz

2018-03-01

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics. © 2017 American Academy of Forensic Sciences.

Should DNA sequence be incorporated with other taxonomical data for routine identifying of plant species?

PubMed

Suesatpanit, Tanakorn; Osathanunkul, Kitisak; Madesis, Panagiotis; Osathanunkul, Maslin

2017-08-31

A variety of plants in Acanthaceae have long been used in traditional Thai ailment and commercialised with significant economic value. Nowadays medicinal plants are sold in processed forms and thus morphological authentication is almost impossible. Full identification requires comparison of the specimen with some authoritative sources, such as a full and accurate description and verification of the species deposited in herbarium. Intake of wrong herbals can cause adverse effects. Identification of both raw materials and end products is therefore needed. Here, the potential of a DNA-based identification method, called Bar-HRM (DNA barcoding coupled with High Resolution Melting analysis), in raw material species identification is investigated. DNA barcode sequences from five regions (matK, rbcL, trnH-psbA spacer region, trnL and ITS2) of Acanthaceae species were retrieved for in silico analysis. Then the specific primer pairs were used in HRM assay to generate unique melting profiles for each plants species. The method allows identification of samples lacking necessary morphological parts. In silico analyses of all five selected regions suggested that ITS2 is the most suitable marker for Bar-HRM in this study. The HRM analysis on dried samples of 16 Acanthaceae medicinal species was then performed using primer pair derived from ITS2 region. 100% discrimination of the tested samples at both genus and species level was observed. However, two samples documented as Clinacanthus nutans and Clinacanthus siamensis were recognised as the same species from the HRM analysis. Further investigation reveals that C. siamensis is now accepted as C. nutans. The results here proved that Bar-HRM is a promising technique in species identification of the studied medicinal plants in Acanthaceae. In addition, molecular biological data is currently used in plant taxonomy and increasingly popular in recent years. Here, DNA barcode sequence data should be incorporated with morphological characters in the species identification.

Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

PubMed Central

Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

2013-01-01

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples. PMID:23577179

Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing.

PubMed

Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

2013-01-01

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.

Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

PubMed

Akika, Reem; Awada, Zainab; Mogharbil, Nahed; Zgheib, Nathalie K

2017-07-01

The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

Improvements in Sensible Heat-Flux Parametrization in the High-Resolution Regional Model (HRM) Through the Modified Treatment of the Roughness Length for Heat

NASA Astrophysics Data System (ADS)

Anurose, T. J.; Subrahamanyam, D. Bala

2013-06-01

We discuss the impact of the differential treatment of the roughness lengths for momentum and heat (z_{0m} and z_{0h}) in the flux parametrization scheme of the high-resolution regional model (HRM) for a heterogeneous terrain centred around Thiruvananthapuram, India (8.5°N, 76.9°E). The magnitudes of sensible heat flux ( H) obtained from HRM simulations using the original parametrization scheme differed drastically from the concurrent in situ observations. With a view to improving the performance of this parametrization scheme, two distinct modifications are incorporated: (1) In the first method, a constant value of 100 is assigned to the z_{0m}/z_{0h} ratio; (2) and in the second approach, this ratio is treated as a function of time. Both these modifications in the HRM model showed significant improvements in the H simulations for Thiruvananthapuram and its adjoining regions. Results obtained from the present study provide a first-ever comparison of H simulations using the modified parametrization scheme in the HRM model with in situ observations for the Indian coastal region, and suggest a differential treatment of z_{0m} and z_{0h} in the flux parametrization scheme.

Hydroponic root mats for wastewater treatment-a review.

PubMed

Chen, Zhongbing; Cuervo, Diego Paredes; Müller, Jochen A; Wiessner, Arndt; Köser, Heinz; Vymazal, Jan; Kästner, Matthias; Kuschk, Peter

2016-08-01

Hydroponic root mats (HRMs) are ecotechnological wastewater treatment systems where aquatic vegetation forms buoyant filters by their dense interwoven roots and rhizomes, sometimes supported by rafts or other floating materials. A preferential hydraulic flow is created in the water zone between the plant root mat and the bottom of the treatment system. When the mat touches the bottom of the water body, such systems can also function as HRM filter; i.e. the hydraulic flow passes directly through the root zone. HRMs have been used for the treatment of various types of polluted water, including domestic wastewater; agricultural effluents; and polluted river, lake, stormwater and groundwater and even acid mine drainage. This article provides an overview on the concept of applying floating HRM and non-floating HRM filters for wastewater treatment. Exemplary performance data are presented, and the advantages and disadvantages of this technology are discussed in comparison to those of ponds, free-floating plant and soil-based constructed wetlands. Finally, suggestions are provided on the preferred scope of application of HRMs.

A comparison of homogeneous equilibrium and relaxation model for CO2 expansion inside the two-phase ejector

NASA Astrophysics Data System (ADS)

Palacz, M.; Haida, M.; Smolka, J.; Nowak, A. J.; Hafner, A.

2016-09-01

In this study, the comparison of the accuracy of the homogeneous equilibrium model (HEM) and homogeneous relaxation model (HRM) is presented. Both models were applied to simulate the CO2 expansion inside the two-phase ejectors. Moreover, the mentioned models were implemented in the robust and efficient computational tool ejectorPL. That tool guarantees the fully automated computational process and the repeatable computations for the various ejector shapes and operating conditions. The simulated motive nozzle mass flow rates were compared to the experimentally measured mass flow rates. That comparison was made for both, HEM and HRM. The results showed the unsatisfying fidelity of the HEM for the operating regimes far from the carbon dioxide critical point. On the other hand, the HRM accuracy for such conditions was slightly higher. The approach presented in this paper, showed the limitation of applicability of both two-phase models for the expansion phenomena inside the ejectors.

High-resolution manometry findings in patients with an intrathoracic stomach.

PubMed

Martinelo, Vanderlei; Mardiros Herbella, Fernando Augusto; Patti, Marco G

2015-04-01

Intrathoracic stomach is a rare finding. The real value of the high-resolution manometry (HRM) in the preoperative evaluation of these patients has not yet being fully tested. This study aims to evaluate: 1) the HRM pattern of patients with an intrathoracic stomach; and 2) HRM findings as predictors for prosthetic reinforcement of the hiatus. We reviewed 33 patients (27 women, mean age 66 years) with an intrathoracic stomach who underwent HRM. Fifteen patients did the HRM as part of preoperative workup and were operated on in our institution. All patients were submitted to a laparoscopic Nissen fundoplication. HRM results show that the lower esophageal sphincter (LES) was transposed in all patients. Hiatal hernia was diagnosed in 21 (63%) patients. The length of the hernia was 4 ± 2 cm (range, 1 to 9 cm). LES oscillation was observed in 23 (69%) patients with a mean of 1 ± 0.4 cm (range, 0.4 to 2 cm). Hiatal mesh reinforcement was necessary in five (33%) of the operated patients. HRM findings did not predict hiatal mesh reinforcement. Our results show that: 1) HRM has a poor sensibility for hiatal hernia diagnosis; 2) half of the patients with an intrathoracic stomach have a normal HRM; and 3) HRM does not predict mesh hiatal hernia repair.

Health worker motivation in Africa: the role of non-financial incentives and human resource management tools.

PubMed

Mathauer, Inke; Imhoff, Ingo

2006-08-29

There is a serious human resource crisis in the health sector in developing countries, particularly in Africa. One of the challenges is the low motivation of health workers. Experience and the evidence suggest that any comprehensive strategy to maximize health worker motivation in a developing country context has to involve a mix of financial and non-financial incentives. This study assesses the role of non-financial incentives for motivation in two cases, in Benin and Kenya. The study design entailed semi-structured qualitative interviews with doctors and nurses from public, private and NGO facilities in rural areas. The selection of health professionals was the result of a layered sampling process. In Benin 62 interviews with health professionals were carried out; in Kenya 37 were obtained. Results from individual interviews were backed up with information from focus group discussions. For further contextual information, interviews with civil servants in the Ministry of Health and at the district level were carried out. The interview material was coded and quantitative data was analysed with SPSS software. The study shows that health workers overall are strongly guided by their professional conscience and similar aspects related to professional ethos. In fact, many health workers are demotivated and frustrated precisely because they are unable to satisfy their professional conscience and impeded in pursuing their vocation due to lack of means and supplies and due to inadequate or inappropriately applied human resources management (HRM) tools. The paper also indicates that even some HRM tools that are applied may adversely affect the motivation of health workers. The findings confirm the starting hypothesis that non-financial incentives and HRM tools play an important role with respect to increasing motivation of health professionals. Adequate HRM tools can uphold and strengthen the professional ethos of doctors and nurses. This entails acknowledging their professionalism and addressing professional goals such as recognition, career development and further qualification. It must be the aim of human resources management/quality management (HRM/QM) to develop the work environment so that health workers are enabled to meet their personal and the organizational goals.

Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

PubMed

Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

2015-04-01

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Association of High-Resolution Manometry Metrics with the Symptoms of Achalasia and the Symptomatic Outcomes of Peroral Esophageal Myotomy

PubMed Central

Tang, Yurong; Xie, Chen; Wang, Meifeng; Jiang, Liuqin; Shi, Ruihua; Lin, Lin

2015-01-01

Background High-resolution manometry (HRM) has improved the accuracy of manometry in detecting achalasia and has helped distinguish between clinically relevant subtypes. This study investigated whether HRM metrics correlate with the achalasia symptoms and symptomatic outcomes of peroral esophageal myotomy (POEM). Methods Of the 30 patients who were enrolled, 25 were treated with POEM, 12 of who underwent HRM after 3 months. All the patients completed the Eckardt score questionnaires, and those who underwent POEM were followed up for about 6 months. Pearson correlation was used to assess the relationship between the HRM metrics and symptoms and outcomes. Key results The integrated relaxation pressure (IRP) score positively correlated with the total Eckardt score, regurgitation score and weight loss score in all the patients, and with the weight loss score in type I achalasia. In 25 patients (10 patients, type I; 15 patients, type II) who underwent POEM, the total Eckardt scores and individual symptom scores significantly decreased after surgery. Changes in the Eckardt scores were similar between type I and type II. Further, the Eckardt scores and weight loss score changes were positively correlated with baseline IRP. Twelve patients (4 patients, type I; 8 patients, type II) underwent HRM again after POEM. IRP changed significantly after POEM, as did the DEP in type II. The IRP changes after POEM were positively correlated with the Eckardt score changes. Conclusions & Inferences IRP is correlated with the symptoms and outcomes of achalasia patients. Thus, HRM is effective for assessing the severity of achalasia and can predict the efficacy of POEM. PMID:26421919

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

PubMed

Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

2018-01-02

Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.

Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

PubMed Central

Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

2013-01-01

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

PubMed Central

Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

2015-01-01

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

PubMed Central

Hanson, Erin K.; Ballantyne, Jack

2014-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence. PMID:24715968

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

PubMed

Hanson, Erin K; Ballantyne, Jack

2013-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.

PubMed

Lochlainn, Seosamh Ó; Amoah, Stephen; Graham, Neil S; Alamer, Khalid; Rios, Juan J; Kurup, Smita; Stoute, Andrew; Hammond, John P; Østergaard, Lars; King, Graham J; White, Phillip J; Broadley, Martin R

2011-12-08

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

PubMed Central

2011-01-01

Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer: a cohort study

PubMed Central

Negru, Serban; Papadopoulou, Eirini; Apessos, Angela; Stanculeanu, Dana Lucia; Ciuleanu, Eliade; Volovat, Constantin; Croitoru, Adina; Kakolyris, Stylianos; Aravantinos, Gerasimos; Ziras, Nikolaos; Athanasiadis, Elias; Touroutoglou, Nikolaos; Pavlidis, Nikolaos; Kalofonos, Haralabos P; Nasioulas, George

2014-01-01

Objectives Treatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations. Setting Diagnostic molecular laboratory located in Athens, Greece. Participants 2425 patients with colorectal cancer participated in the study. Primary and secondary outcome measures 2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing. Results KRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method. Conclusions The HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer. PMID:24859998

Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance

PubMed Central

Wahyuningsih, Hesty; K Cayami, Ferdy; Bahrudin, Udin; A Sobirin, Mochamad; EP Mundhofir, Farmaditya; MH Faradz, Sultana; Hisatome, Ichiro

2017-01-01

Background High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. Methods Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. Results Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100–400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. Conclusion In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1. PMID:28331418

Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region▿

PubMed Central

Steer, Penelope A.; Kirkpatrick, Naomi C.; O'Rourke, Denise; Noormohammadi, Amir H.

2009-01-01

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens. PMID:19036935

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis.

PubMed

Papavasileiou, Antonios; Madesis, Panagiotis B; Karaoglanidis, George S

2016-09-01

Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit.

High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar.

PubMed

Croxford, Adam E; Rogers, Tom; Caligari, Peter D S; Wilkinson, Michael J

2008-01-01

* The provision of sequence-tagged site (STS) anchor points allows meaningful comparisons between mapping studies but can be a time-consuming process for nonmodel species or orphan crops. * Here, the first use of high-resolution melt analysis (HRM) to generate STS markers for use in linkage mapping is described. This strategy is rapid and low-cost, and circumvents the need for labelled primers or amplicon fractionation. * Using white lupin (Lupinus albus, x = 25) as a case study, HRM analysis was applied to identify 91 polymorphic markers from expressed sequence tag (EST)-derived and genomic libraries. Of these, 77 generated STS anchor points in the first fully resolved linkage map of the species. The map also included 230 amplified fragment length polymorphisms (AFLP) loci, spanned 1916 cM (84.2% coverage) and divided into the expected 25 linkage groups. * Quantitative trait loci (QTL) analyses performed on the population revealed genomic regions associated with several traits, including the agronomically important time to flowering (tf), alkaloid synthesis and stem height (Ph). Use of HRM-STS markers also allowed us to make direct comparisons between our map and that of the related crop, Lupinus angustifolius, based on the conversion of RFLP, microsatellite and single nucleotide polymorphism (SNP) markers into HRM markers.

Butyrylated starch intake can prevent red meat-induced O6-methyl-2-deoxyguanosine adducts in human rectal tissue: a randomised clinical trial.

PubMed

Le Leu, Richard K; Winter, Jean M; Christophersen, Claus T; Young, Graeme P; Humphreys, Karen J; Hu, Ying; Gratz, Silvia W; Miller, Rosalind B; Topping, David L; Bird, Anthony R; Conlon, Michael A

2015-07-01

Epidemiological studies have identified increased colorectal cancer (CRC) risk with high red meat (HRM) intakes, whereas dietary fibre intake appears to be protective. In the present study, we examined whether a HRM diet increased rectal O(6)-methyl-2-deoxyguanosine (O(6)MeG) adduct levels in healthy human subjects, and whether butyrylated high-amylose maize starch (HAMSB) was protective. A group of twenty-three individuals consumed 300 g/d of cooked red meat without (HRM diet) or with 40 g/d of HAMSB (HRM+HAMSB diet) over 4-week periods separated by a 4-week washout in a randomised cross-over design. Stool and rectal biopsy samples were collected for biochemical, microbial and immunohistochemical analyses at baseline and at the end of each 4-week intervention period. The HRM diet increased rectal O(6)MeG adducts relative to its baseline by 21% (P < 0.01), whereas the addition of HAMSB to the HRM diet prevented this increase. Epithelial proliferation increased with both the HRM (P < 0.001) and HRM + HAMSB (P < 0.05) diets when compared with their respective baseline levels, but was lower following the HRM + HAMSB diet compared with the HRM diet (P < 0.05). Relative to its baseline, the HRM + HAMSB diet increased the excretion of SCFA by over 20% (P < 0.05) and increased the absolute abundances of the Clostridium coccoides group (P < 0.05), the Clostridium leptum group (P < 0.05), Lactobacillus spp. (P < 0.01), Parabacteroides distasonis (P < 0.001) and Ruminococcus bromii (P < 0.05), but lowered Ruminococcus torques (P < 0.05) and the proportions of Ruminococcus gnavus, Ruminococcus torques and Escherichia coli (P < 0.01). HRM consumption could increase the risk of CRC through increased formation of colorectal epithelial O(6)MeG adducts. HAMSB consumption prevented red meat-induced adduct formation, which may be associated with increased stool SCFA levels and/or changes in the microbiota composition.

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

PubMed Central

Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

2013-01-01

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula. PMID:23825624

Rapid detection and simultaneous genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula using real-time PCR and high resolution melting (HRM) analysis.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

2013-01-01

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.

Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product.

PubMed

Buddhachat, Kittisak; Osathanunkul, Maslin; Madesis, Panagiotis; Chomdej, Siriwadee; Ongchai, Siriwan

2015-11-15

The Phyllanthus genus, a plant used in traditional Thai medicine, has according to several pharmacopeias hepatoprotective properties. Not only is the anatomical morphology of these species relatively similar but they also share the Thai common names Look-Tai-Bai (ลูกใต้ใบ) and Yah-Tai-Bai (หญ้าใต้ใบ), which might cause confusion for laypersons. This study attempted to develop a method for accurate identification of Phyllanthus species, especially Phyllanthus amarus, and to detect contaminants in P. amarus products by using DNA barcoding coupled with high resolution melting (HRM) analysis (bar-HRM). Two plastid loci (rbcL and trnL) were chosen for DNA barcoding to generate a suitable primer for distinguishing Phyllanthus species by HRM analysis. The five species of Phyllanthus were subjected to amplification for testing the specificity and discrimination power of the designed primers derived from rbcL and trnL regions. Sensitivity of the method (DNA barcoding conjugated with HRM) to detect adulterant in P. amarus samples was evaluated. The commercial P. amarus products obtained from a local market were authenticated. The primer pair derived from trnL DNA barcoding (PhylltrnL) had more specificity and power of discrimination for Phyllanthus species than that derived from rbcL DNA barcoding (PhyllrbcL). The result showed that Tm of P. amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus airy-shawii, and Phyllanthus virgatus was 74.3±0.08, 73.04±0.07, 73.36±0.05, 72.21±0.06, 72.77±0.15°C, respectively. This method proved to be a very sensitive tool that can be used for rapid detection of contamination as low as 1% of other Phyllanthus species in P. amarus admixtures. All commercial products of P. amarus obtained from a local market in Thailand were found to contain pure raw materials of P. amarus without any substitution or contamination. Our results indicated that the use of DNA barcoding coupled with HRM was an efficient molecular tool for correct species identification. This molecular tool provides a noteworthy benefit for quality control of medicinal plants and industry plants for pharmacological prospects. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Validity of a heart rate monitor during work in the laboratory and on the Space Shuttle

NASA Technical Reports Server (NTRS)

Moore, A. D. Jr; Lee, S. M.; Greenisen, M. C.; Bishop, P.

1997-01-01

Accurate heart rate measurement during work is required for many industrial hygiene and ergonomics situations. The purpose of this investigation was to determine the validity of heart rate measurements obtained by a simple, lightweight, commercially available wrist-worn heart rate monitor (HRM) during work (cycle exercise) sessions conducted in the laboratory and also during the particularly challenging work environment of space flight. Three different comparisons were made. The first compared HRM data to simultaneous electrocardiogram (ECG) recordings of varying heart rates that were generated by an ECG simulator. The second compared HRM data to ECG recordings collected during work sessions of 14 subjects in the laboratory. Finally, ECG downlink and HRM data were compared in four astronauts who performed cycle exercise during space flight. The data were analyzed using regression techniques. The results were that the HRM recorded virtually identical heart rates compared with ECG recordings for the data set generated by an ECG simulator. The regression equation for the relationship between ECG versus HRM heart rate data during work in the laboratory was: ECG HR = 0.99 x (HRM) + 0.82 (r2 = 0.99). Finally, the agreement between ECG downlink data and HRM data during space flight was also very high, with the regression equation being: Downlink ECG HR = 1.05 x (HRM) -5.71 (r2 = 0.99). The results of this study indicate that the HRM provides accurate data and may be used to reliably obtain valid data regarding heart rate responses during work.

Lactase persistence genotyping on whole blood by loop-mediated isothermal amplification and melting curve analysis.

PubMed

Abildgaard, Anders; Tovbjerg, Sara K; Giltay, Axel; Detemmerman, Liselot; Nissen, Peter H

2018-03-26

The lactase persistence phenotype is controlled by a regulatory enhancer region upstream of the Lactase (LCT) gene. In northern Europe, specifically the -13910C > T variant has been associated with lactase persistence whereas other persistence variants, e.g. -13907C > G and -13915 T > G, have been identified in Africa and the Middle East. The aim of the present study was to compare a previously developed high resolution melting assay (HRM) with a novel method based on loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) with both whole blood and DNA as input material. To evaluate the LAMP-MC method, we used 100 whole blood samples and 93 DNA samples in a two tiered study. First, we studied the ability of the LAMP-MC method to produce specific melting curves for several variants of the LCT enhancer region. Next, we performed a blinded comparison between the LAMP-MC method and our existing HRM method with clinical samples of unknown genotype. The LAMP-MC method produced specific melting curves for the variants at position -13909, -13910, -13913 whereas the -13907C > G and -13915 T > G variants produced indistinguishable melting profiles. The LAMP-MC assay is a simple method for lactase persistence genotyping and compares well with our existing HRM method. Copyright © 2018. Published by Elsevier B.V.

Esophageal symptoms and their lack of association with high-resolution manometry in systemic sclerosis patients.

PubMed

Arana-Guajardo, Ana Cecilia; Barrera-Torres, Gustavo; Villarreal-Alarcón, Miguel Ángel; Vega-Morales, David; Esquivel-Valerio, Jorge Antonio

2017-12-16

The esophageal involvement in systemic sclerosis (SSc) causes impact in the morbidity and mortality. High resolution manometry assesses esophageal involvement. Our aim was to categorize esophageal motor disorder in patients with SSc by HRM. We carried out an observational, descriptive and cross-sectional study. All patients underwent HRM as well as semi-structured interviews to assess frequency and severity of upper GI symptoms. Patients also completed the gastroesophageal reflux questionnaire (Carlsson-Dent). We included 19 patients with SSc, 1 with morphea, and 1 with scleroderma sine scleroderma. Dysphagia and heartburn were the most frequent symptoms (61% each). We found an abnormal HRM in 15 (71.4%) patients. We found no statistically significant association between clinical or demographic variables and an abnormal HRM, or between any upper GI symptom and HRM findings. We observed a high prevalence of esophageal symptoms and of HRM abnormalities. However, there was no clear association between symptomatology and HRM findings. HRM does not seem to accurately predict upper GI symptomatology. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Human resource management in post-conflict health systems: review of research and knowledge gaps.

PubMed

Roome, Edward; Raven, Joanna; Martineau, Tim

2014-01-01

In post-conflict settings, severe disruption to health systems invariably leaves populations at high risk of disease and in greater need of health provision than more stable resource-poor countries. The health workforce is often a direct victim of conflict. Effective human resource management (HRM) strategies and policies are critical to addressing the systemic effects of conflict on the health workforce such as flight of human capital, mismatches between skills and service needs, breakdown of pre-service training, and lack of human resource data. This paper reviews published literatures across three functional areas of HRM in post-conflict settings: workforce supply, workforce distribution, and workforce performance. We searched published literatures for articles published in English between 2003 and 2013. The search used context-specific keywords (e.g. post-conflict, reconstruction) in combination with topic-related keywords based on an analytical framework containing the three functional areas of HRM (supply, distribution, and performance) and several corresponding HRM topic areas under these. In addition, the framework includes a number of cross-cutting topics such as leadership and governance, finance, and gender. The literature is growing but still limited. Many publications have focused on health workforce supply issues, including pre-service education and training, pay, and recruitment. Less is known about workforce distribution, especially governance and administrative systems for deployment and incentive policies to redress geographical workforce imbalances. Apart from in-service training, workforce performance is particularly under-researched in the areas of performance-based incentives, management and supervision, work organisation and job design, and performance appraisal. Research is largely on HRM in the early post-conflict period and has relied on secondary data. More primary research is needed across the areas of workforce supply, workforce distribution, and workforce performance. However, this should apply a longer-term focus throughout the different post-conflict phases, while paying attention to key cross-cutting themes such as leadership and governance, gender equity, and task shifting. The research gaps identified should enable future studies to examine how HRM could be used to meet both short and long term objectives for rebuilding health workforces and thereby contribute to achieving more equitable and sustainable health systems outcomes after conflict.

High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples.

PubMed

Chelbi, Hanen; Essid, Rym; Jelassi, Refka; Bouzekri, Nesrine; Zidi, Ines; Ben Salah, Hamza; Mrad, Ilhem; Ben Sghaier, Ines; Abdelmalek, Rym; Aissa, Sameh; Bouratbine, Aida; Aoun, Karim

2018-02-01

Cryptosporidiosis represents a major public health problem. This infection, caused by a protozoan parasite of the genus Cryptosporidium, has been reported worldwide as a frequent cause of diarrhoea. In the immunocompetent host, the typical watery diarrhea can be self-limiting. However, it is severe and chronic, in the immunocompromised host and may cause death. Cryptosporidium spp. are coccidians, which complete their life cycle in both humans and animals. The two species C. hominis and C. parvum are the major cause of human infection. Compared to studies on C. hominis and C. parvum, only a few studies have developed methods to identify C. meleagridis. To develop a new real time PCR-coupled High resolution melting assay allowing the detection for C. meleagridis, in addition of the other dominant species (C. hominis and C. parvum). The polymorphic sequence on the dihydrofolate reductase gene (DHFR) of three species was sequenced to design primers pair and establish a sensitive real-time PCR coupled to a high-resolution melting-curve (HRM) analysis method, allowing the detection of Cryptosporidium sp. and discrimination between three prevalent species in Tunisia. We analyzed a collection of 42 archived human isolates of the three studied species. Real-time PCR coupled to HRM assay allowed detection of Cryptosporidium, using the new designed primers, and basing on melting profile, we can distinguish C. meleagridis species in addition to C. parvum and C. hominis. We developed a qPCR-HRM assay that allows Cryptosporidium genotyping. This method is sensitive and able to distinguish three Cryptosporidium species. Copyright © 2017. Published by Elsevier Ltd.

What difference does ("good") HRM make?

PubMed

Buchan, James

2004-06-07

The importance of human resources management (HRM) to the success or failure of health system performance has, until recently, been generally overlooked. In recent years it has been increasingly recognised that getting HR policy and management "right" has to be at the core of any sustainable solution to health system performance. In comparison to the evidence base on health care reform-related issues of health system finance and appropriate purchaser/provider incentive structures, there is very limited information on the HRM dimension or its impact.Despite the limited, but growing, evidence base on the impact of HRM on organisational performance in other sectors, there have been relatively few attempts to assess the implications of this evidence for the health sector. This paper examines this broader evidence base on HRM in other sectors and examines some of the underlying issues related to "good" HRM in the health sector.The paper considers how human resource management (HRM) has been defined and evaluated in other sectors. Essentially there are two sub-themes: how have HRM interventions been defined? and how have the effects of these interventions been measured in order to identify which interventions are most effective? In other words, what is "good" HRM?The paper argues that it is not only the organisational context that differentiates the health sector from many other sectors, in terms of HRM. Many of the measures of organisational performance are also unique. "Performance" in the health sector can be fully assessed only by means of indicators that are sector-specific. These can focus on measures of clinical activity or workload (e.g. staff per occupied bed, or patient acuity measures), on measures of output (e.g. number of patients treated) or, less frequently, on measures of outcome (e.g. mortality rates or rate of post-surgery complications).The paper also stresses the need for a "fit" between the HRM approach and the organisational characteristics, context and priorities, and for recognition that so-called "bundles" of linked and coordinated HRM interventions will be more likely to achieve sustained improvements in organisational performance than single or uncoordinated interventions.

UGT1A1 (TA)n genotyping in sickle-cell disease: high resolution melting (HRM) curve analysis or direct sequencing, what is the best way?

PubMed

Thomas, Vincent; Mazard, Blandine; Garcia, Caroline; Lacan, Philippe; Gagnieu, Marie-Claude; Joly, Philippe

2013-09-23

Minucci et al. have proposed in 2010 a rapid, simple and cost-effective HRM method on the LightCycler 480® apparatus (Roche) for the determination of the 6/6, 6/7 and 7/7 genotypes of the (TA)n UGT1A1 promoter polymorphism. However, they have not studied the n=5 and n=8 alleles which can be quite frequent in sickle-cell disease patients. The aim of our study was to test this HRM protocol to all the 10 possible (TA)n UGT1A1 genotypes (i.e. 5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8 and 8/8) by using our SCD cohort of patients. All genotypes could be unambiguously identified except 6/7 and 6/8 which give a similar HRM profile. For those two genotypes, the differentiation necessitates either a direct Sanger sequencing or a second PCR protocol followed by a 3% agarose gel migration. For the (TA)n UGT1A1 promoter genotyping of African patients, each lab has to wonder what is the best way between (i) direct Sanger sequencing of all patients and (ii) HRM protocol for all patients followed by a complementary analysis to differentiate the 6/7 and 6/8 genotypes. © 2013. Published by Elsevier B.V. All rights reserved.

Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

PubMed

Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

2015-08-01

HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functions within the Naval Air Training Command

DTIC Science & Technology

1984-02-15

Resource Management < HRM ), Leadership and Management Education and Training (LMET), Officer and Enlisted Career Counseling and Retention programs...as division officer for assigned HRM and career counselor personnel. i3i/byQ]§Q_BesDurce_rianaaement_Seeci.ali.5t N13-5 Advises and assists Code...13 in the development, evaluation and standardization of NATRACOM HRM programs. N13-6 Responsible for monitoring NAVAVSCOLSCOM HRM Support Team

Application of high-resolution melting analysis for authenticity testing of valuable Dendrobium commercial products.

PubMed

Dong, Xiaoman; Jiang, Chao; Yuan, Yuan; Peng, Daiyin; Luo, Yuqin; Zhao, Yuyang; Huang, Luqi

2018-01-01

The accurate identification of botanical origin in commercial products is important to ensure food authenticity and safety for consumers. The Dendrobium species have long been commercialised as functional food supplements and herbal medicines in Asia. Three valuable Dendrobium species, namely Dendrobium officinale, D. huoshanense and D. moniliforme, are often mutually adulterated in trade products in pursuit of higher profit. In this paper, a rapid and reliable semi-quantitative method for identifying the botanical origin of Dendrobium products in terminal markets was developed using high-resolution melting (HRM) analysis with specific primer pairs to target the trnL-F region. The HRM analysis method detected amounts of D. moniliforme adulterants as low as 1% in D. huoshanense or D. officinale products. The results have demonstrated that HRM analysis is a fast and effective tool for the differentiation of these Dendrobium species both for their authenticity as well as for the semi-quantitative determination of the purity of their processed products. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Application of the SYBR Green real-time HRM PCR technique in the differentiation of the Babesia canis canis protozoa isolated in the areas of eastern Poland.

PubMed

Adaszek, Lukasz; Winiarczyk, Stanisław

2010-04-01

The aim of this study was to determine the usefulness of the real-time polymerised chain reaction (PCR) high-resolution melting (HRM) method in the differentiation of the Babesia canis canis protozoa isolated from dogs in the areas of eastern Poland. The studies involved 20 isolates of B. canis canis qualified depending on the analysis of the 18S RNA gene sequence to group A (EU 622792) and 20 isolates qualified to group B (EU 622793). It was proven with the real-time PCR technique that the melting temperature (Tm) of the obtained products of amplification was 78 degrees C for the representatives of group A and 81 degrees C for the representatives of group B, which proves that the real-time SYBR Green HRM PCR method is a technique allowing for the differentiation of the B. canis isolates which are slightly different with respect to the genetic structure, without the necessity to carry out time-consuming studies, i.e., sequencing and restriction fragment length polymorphism.

The Chronic Effects of Low- and High-Intensity Resistance Training on Muscular Fitness in Adolescents

PubMed Central

Assunção, Ari R.; Bottaro, Martim; Ferreira-Junior, João B.; Izquierdo, Mikel; Cadore, Eduardo L.; Gentil, Paulo

2016-01-01

To compare the effects of high-load, low-repetition maximum (LRM) and low-load, high-repetition maximum (HRM) resistance training regimens on muscular fitness in untrained adolescents. Forty-five untrained adolescents of both sexes (13.7±0.8 years; 161.3±7.5 cm, 56.8±13.4 kg) were randomly assigned into one of three groups: 1) LRM (n = 17): volunteers performed three sets of 4-6-repetition maximum (RM); 2) HRM (n = 16): volunteers performed three sets of 12–15 RM; and 3) control (CON, n = 12). Training was performed two times a week for 9 weeks. After training, there were significant increases in 1 RM chest press (LRM = 14.8% and HRM = 14.2%, p<0.05) and squat (LRM = 26.4% and HRM = 25.7%, p<0.05), with no differences between the LRM and HRM groups (p>0.05). Additionally, muscular endurance increased significantly for the chest press (LRM = 14.5% and HRM = 21.8%, p<0.05) and squat test (LRM = 31.4% and HRM = 32.4%, p<0.05) following resistance training, with no difference between the LRM and HRM groups (p>0.05). These results suggest that both high-load, low-repetition and moderate-load, high-repetition resistance training can be prescribed to improve muscular fitness in untrained adolescents. PMID:27509050

Lack of correlation between HRM metrics and symptoms during the manometric protocol.

PubMed

Xiao, Yinglian; Kahrilas, Peter J; Nicodème, Frédéric; Lin, Zhiyue; Roman, Sabine; Pandolfino, John E

2014-04-01

Although esophageal motor disorders are associated with chest pain and dysphagia, minimal data support a direct relationship between abnormal motor function and symptoms. This study investigated whether high-resolution manometry (HRM) metrics correlate with symptoms. Consecutive HRM patients without previous surgery were enrolled. HRM studies included 10 supine liquid, 5 upright liquid, 2 upright viscous, and 2 upright solid swallows. All patients evaluated their esophageal symptom for each upright swallow. Symptoms were graded on a 4-point likert score (0, none; 1, mild; 2, moderate; 3, severe). The individual liquid, viscous or solid upright swallow with the maximal symptom score was selected for analysis in each patient. HRM metrics were compared between groups with and without symptoms during the upright liquid protocol and the provocative protocols separately. A total of 269 patients recorded symptoms during the upright liquid swallows and 72 patients had a swallow symptom score of 1 or greater. Of the 269 patients, 116 recorded symptoms during viscous or solid swallows. HRM metrics were similar between swallows with and without associated symptoms in the upright, viscous, and solid swallows. No correlation was noted between HRM metrics and symptom scores among swallow types. Esophageal symptoms are not related to abnormal motor function defined by HRM during liquid, viscous or solid bolus swallows in the upright position. Other factors beyond circular muscle contraction patterns should be explored as possible causes of symptom generation.

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

Projecting Individualized Absolute Invasive Breast Cancer Risk in US Hispanic Women

PubMed Central

John, Esther M.; Slattery, Martha L.; Gomez, Scarlett Lin; Yu, Mandi; LaCroix, Andrea Z.; Pee, David; Chlebowski, Rowan T.; Hines, Lisa M.; Thompson, Cynthia A.; Gail, Mitchell H.

2017-01-01

Background: There is no model to estimate absolute invasive breast cancer risk for Hispanic women. Methods: The San Francisco Bay Area Breast Cancer Study (SFBCS) provided data on Hispanic breast cancer case patients (533 US-born, 553 foreign-born) and control participants (464 US-born, 947 foreign-born). These data yielded estimates of relative risk (RR) and attributable risk (AR) separately for US-born and foreign-born women. Nativity-specific absolute risks were estimated by combining RR and AR information with nativity-specific invasive breast cancer incidence and competing mortality rates from the California Cancer Registry and Surveillance, Epidemiology, and End Results program to develop the Hispanic risk model (HRM). In independent data, we assessed model calibration through observed/expected (O/E) ratios, and we estimated discriminatory accuracy with the area under the receiver operating characteristic curve (AUC) statistic. Results: The US-born HRM included age at first full-term pregnancy, biopsy for benign breast disease, and family history of breast cancer; the foreign-born HRM also included age at menarche. The HRM estimated lower risks than the National Cancer Institute’s Breast Cancer Risk Assessment Tool (BCRAT) for US-born Hispanic women, but higher risks in foreign-born women. In independent data from the Women’s Health Initiative, the HRM was well calibrated for US-born women (observed/expected [O/E] ratio = 1.07, 95% confidence interval [CI] = 0.81 to 1.40), but seemed to overestimate risk in foreign-born women (O/E ratio = 0.66, 95% CI = 0.41 to 1.07). The AUC was 0.564 (95% CI = 0.485 to 0.644) for US-born and 0.625 (95% CI = 0.487 to 0.764) for foreign-born women. Conclusions: The HRM is the first absolute risk model that is based entirely on data specific to Hispanic women by nativity. Further studies in Hispanic women are warranted to evaluate its validity. PMID:28003316

Facilitating job retention for chronically ill employees: perspectives of line managers and human resource managers.

PubMed

Haafkens, Joke A; Kopnina, Helen; Meerman, Martha G M; van Dijk, Frank J H

2011-05-17

Chronic diseases are a leading contributor to work disability and job loss in Europe. Recent EU policies aim to improve job retention among chronically ill employees. Disability and occupational health researchers argue that this requires a coordinated and pro-active approach at the workplace by occupational health professionals, line managers (LMs) and human resource managers (HRM). Little is known about the perspectives of LMs an HRM on what is needed to facilitate job retention among chronically ill employees. The aim of this qualitative study was to explore and compare the perspectives of Dutch LMs and HRM on this issue. Concept mapping methodology was used to elicit and map statements (ideas) from 10 LMs and 17 HRM about what is needed to ensure continued employment for chronically ill employees. Study participants were recruited through a higher education and an occupational health services organization. Participants generated 35 statements. Each group (LMs and HRM) sorted these statements into six thematic clusters. LMs and HRM identified four similar clusters: LMs and HRM must be knowledgeable about the impact of chronic disease on the employee; employees must accept responsibility for work retention; work adaptations must be implemented; and clear company policy. Thematic clusters identified only by LMs were: good manager/employee cooperation and knowledge transfer within the company. Unique clusters identified by HRM were: company culture and organizational support. There were both similarities and differences between the views of LMs and HRM on what may facilitate job retention for chronically ill employees. LMs perceived manager/employee cooperation as the most important mechanism for enabling continued employment for these employees. HRM perceived organizational policy and culture as the most important mechanism. The findings provide information about topics that occupational health researchers and planners should address in developing job retention programs for chronically ill workers.

Esophagogastric Junction pressure morphology: comparison between a station pull-through and real-time 3D-HRM representation.

PubMed

Nicodème, F; Lin, Z; Pandolfino, J E; Kahrilas, P J

2013-09-01

Esophagogastric junction (EGJ) competence is the fundamental defense against reflux making it of great clinical significance. However, characterizing EGJ competence with conventional manometric methodologies has been confounded by its anatomic and physiological complexity. Recent technological advances in miniaturization and electronics have led to the development of a novel device that may overcome these challenges. Nine volunteer subjects were studied with a novel 3D-HRM device providing 7.5 mm axial and 45° radial pressure resolution within the EGJ. Real-time measurements were made at rest and compared to simulations of a conventional pull-through made with the same device. Moreover, 3D-HRM recordings were analyzed to differentiate contributing pressure signals within the EGJ attributable to lower esophageal sphincter (LES), diaphragm, and vasculature. 3D-HRM recordings suggested that sphincter length assessed by a pull-through method greatly exaggerated the estimate of LES length by failing to discriminate among circumferential contractile pressure and asymmetric extrinsic pressure signals attributable to diaphragmatic and vascular structures. Real-time 3D EGJ recordings found that the dominant constituents of EGJ pressure at rest were attributable to the diaphragm. 3D-HRM permits real-time recording of EGJ pressure morphology facilitating analysis of the EGJ constituents responsible for its function as a reflux barrier making it a promising tool in the study of GERD pathophysiology. The enhanced axial and radial recording resolution of the device should facilitate further studies to explore perturbations in the physiological constituents of EGJ pressure in health and disease. © 2013 John Wiley & Sons Ltd.

The high resolution melting analysis (HRM) as a molecular tool for monitoring parasites of the wildlife.

PubMed

Héritier, Laurent; Verneau, Olivier; Breuil, Gregory; Meistertzheim, Anne-Leila

2017-04-01

In an interconnected world, the international pet trade on wild animals is becoming increasingly important. As a consequence, non-native parasite species are introduced, which affect the health of wildlife and contribute to the loss of biodiversity. Because the investigation of parasite diversity within vulnerable host species implies the molecular identification of large samples of parasite eggs, the sequencing of DNA barcodes is time-consuming and costly. Thereby, the objectives of our study were to apply the high resolution melting (HRM) approach for species determination from pools of parasite eggs. Molecular assays were validated on flatworm parasites (polystomes) infecting the Mediterranean pond turtle Mauremys leprosa and the invasive red-eared slider Trachemys scripta elegans in French natural environments. HRM analysis results indicated that double or multiple parasitic infections could be detected from wild animal populations. They also showed that the cycle of parasite eggs production was not regular over time and may depend on several factors, among which the ecological niche and the target species. Thereby, monitoring parasites from wild endangered animals implies periodic parasitological surveys to avoid false negative diagnostics, based solely on eggs production.

Clinical Application of Esophageal High-resolution Manometry in the Diagnosis of Esophageal Motility Disorders.

PubMed

van Hoeij, Froukje B; Bredenoord, Albert J

2016-01-31

Esophageal high-resolution manometry (HRM) is replacing conventional manometry in the clinical evaluation of patients with esophageal symptoms, especially dysphagia. The introduction of HRM gave rise to new objective metrics and recognizable patterns of esophageal motor function, requiring a new classification scheme: the Chicago classification. HRM measurements are more detailed and more easily performed compared to conventional manometry. The visual presentation of acquired data improved the analysis and interpretation of esophageal motor function. This led to a more sensitive, accurate, and objective analysis of esophageal motility. In this review we discuss how HRM changed the way we define and categorize esophageal motility disorders. Moreover, we discuss the clinical applications of HRM for each esophageal motility disorder separately.

Clinical Application of Esophageal High-resolution Manometry in the Diagnosis of Esophageal Motility Disorders

PubMed Central

van Hoeij, Froukje B; Bredenoord, Albert J

2016-01-01

Esophageal high-resolution manometry (HRM) is replacing conventional manometry in the clinical evaluation of patients with esophageal symptoms, especially dysphagia. The introduction of HRM gave rise to new objective metrics and recognizable patterns of esophageal motor function, requiring a new classification scheme: the Chicago classification. HRM measurements are more detailed and more easily performed compared to conventional manometry. The visual presentation of acquired data improved the analysis and interpretation of esophageal motor function. This led to a more sensitive, accurate, and objective analysis of esophageal motility. In this review we discuss how HRM changed the way we define and categorize esophageal motility disorders. Moreover, we discuss the clinical applications of HRM for each esophageal motility disorder separately. PMID:26631942

Human resource management in post-conflict health systems: review of research and knowledge gaps

PubMed Central

2014-01-01

In post-conflict settings, severe disruption to health systems invariably leaves populations at high risk of disease and in greater need of health provision than more stable resource-poor countries. The health workforce is often a direct victim of conflict. Effective human resource management (HRM) strategies and policies are critical to addressing the systemic effects of conflict on the health workforce such as flight of human capital, mismatches between skills and service needs, breakdown of pre-service training, and lack of human resource data. This paper reviews published literatures across three functional areas of HRM in post-conflict settings: workforce supply, workforce distribution, and workforce performance. We searched published literatures for articles published in English between 2003 and 2013. The search used context-specific keywords (e.g. post-conflict, reconstruction) in combination with topic-related keywords based on an analytical framework containing the three functional areas of HRM (supply, distribution, and performance) and several corresponding HRM topic areas under these. In addition, the framework includes a number of cross-cutting topics such as leadership and governance, finance, and gender. The literature is growing but still limited. Many publications have focused on health workforce supply issues, including pre-service education and training, pay, and recruitment. Less is known about workforce distribution, especially governance and administrative systems for deployment and incentive policies to redress geographical workforce imbalances. Apart from in-service training, workforce performance is particularly under-researched in the areas of performance-based incentives, management and supervision, work organisation and job design, and performance appraisal. Research is largely on HRM in the early post-conflict period and has relied on secondary data. More primary research is needed across the areas of workforce supply, workforce distribution, and workforce performance. However, this should apply a longer-term focus throughout the different post-conflict phases, while paying attention to key cross-cutting themes such as leadership and governance, gender equity, and task shifting. The research gaps identified should enable future studies to examine how HRM could be used to meet both short and long term objectives for rebuilding health workforces and thereby contribute to achieving more equitable and sustainable health systems outcomes after conflict. PMID:25295071

Genotyping of Listeria monocytogenes isolates from poultry carcasses using high resolution melting (HRM) analysis.

PubMed

Sakaridis, Ioannis; Ganopoulos, Ioannis; Madesis, Panagiotis; Tsaftaris, Athanasios; Argiriou, Anagnostis

2014-01-02

An outbreak situation of human listeriosis requires a fast and accurate protocol for typing Listeria monocytogenes . Existing techniques are either characterized by low discriminatory power or are laborious and require several days to give a final result. Polymerase chain reaction (PCR) coupled with high resolution melting (HRM) analysis was investigated in this study as an alternative tool for a rapid and precise genotyping of L. monocytogenes isolates. Fifty-five isolates of L. monocytogenes isolated from poultry carcasses and the environment of four slaughterhouses were typed by HRM analysis using two specific markers, internalin B and ssrA genes. The analysis of genotype confidence percentage of L. monocytogenes isolates produced by HRM analysis generated dendrograms with two major groups and several subgroups. Furthermore, the analysis of the HRM curves revealed that all L. monocytogenes isolates could easily be distinguished. In conclusion, HRM was proven to be a fast and powerful tool for genotyping isolates of L. monocytogenes .

Buffers or Boosters? The Role of HRM Practices in Older Workers' Experience of Stereotype Threat.

PubMed

Oliveira, Eduardo André da Silva; Cabral-Cardoso, Carlos José

2018-01-02

Building on the social identity approach and the HRM literature, this two-wave cross-sectional study examined the effects of negative age-based metastereotypes on the age-based stereotype threat experience of older workers and on organizational disidentification. The moderator role of HRM practices in the relationship between negative age-based metastereotypes and age-based stereotype threat was also investigated. Older workers survey results (n = 469) from 14 manufacturing companies indicated that negative age-based metastereotypes correlate positively with stereotype threat. Moderation results showed that age-awareness HRM practices (training for older workers) reinforced age-based stereotype threat, whereas general HRM practices (recognition and respect) impaired it. The article suggests that the combination of age-based metastereotypes with a stereotype threat framework contributes to further understand older workers' beliefs and attitudes. It also indicates that to be effective, HRM practices should emphasize positive social identities older workers share with their colleagues, rather than giving older workers special treatment that may, after all, reinforce stigmatization.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting.

PubMed

Matsumoto, Nariyoshi; Mori, Sayaka; Hasegawa, Hiroo; Sasaki, Daisuke; Mori, Hayato; Tsuruda, Kazuto; Imanishi, Daisuke; Imaizumi, Yoshitaka; Hata, Tomoko; Kaku, Norihito; Kosai, Kousuke; Uno, Naoki; Miyazaki, Yasushi; Yanagihara, Katsunori

2016-11-01

Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60-80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). Among 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. Our novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

High resolution melting analysis to genotype the most common variants in the HFE gene.

PubMed

Marotta, Roberta V; Turri, Olivia; Morandi, Antonella; Murano, Manuela; d'Eril, Gianlodovico Melzi; Biondi, Maria Luisa

2011-09-01

High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis. Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls. One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects. HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

PubMed

Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

2012-02-18

As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

Exploring the Best HRM Practices-Performance Relationship: An Empirical Approach

ERIC Educational Resources Information Center

Theriou, George N.; Chatzoglou, Prodromos D.

2009-01-01

Purpose: This paper aims to empirically examine the linkages between best human resource (HRM) practices, knowledge management, organisational learning, organisational capabilities and organisational performance. The proposed framework and findings intend to add to the understanding of the specific processes that mediate between best HRM practices…

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories. PMID:21637859

Ownership and Use of Commercial Physical Activity Trackers Among Finnish Adolescents: Cross-Sectional Study

PubMed Central

Tynjälä, Jorma; Kokko, Sami

2017-01-01

Background Mobile phone apps for monitoring and promoting physical activity (PA) are extremely popular among adults. Devices, such as heart rate monitors or sports watches (HRMs/SWs) that work with these apps are at sufficiently low costs to be available through the commercial markets. Studies have reported an increase in PA levels among adults with devices; however, it is unknown whether the phenomena are similar during early adolescence. At a time when adolescents start to develop their own sense of independence and build friendship, the ease of smartphone availability in developed countries needs to be investigated in important health promoting behaviors such as PA. Objective The objective of this study was to investigate the ownership and usage of PA trackers (apps and HRM/SW) among adolescents in a national representative sample and to examine the association between use of devices and PA levels. Methods The Finnish school-aged physical activity (SPA) study consisted of 4575 adolescents, aged 11-, 13-, and 15-years, who took part in a web-based questionnaire during school time about PA behaviors between April and May 2016. Binary logistic regression analyses were used to test the associations between moderate to vigorous physical activity (MVPA) and devices, after controlling for gender, age, disability, and family affluence. Results PA tracking devices have been categorized into two types, which are accessible to adolescents: (1) apps and (2) HRM/SW. Half the adolescents (2351/4467; 52.63%) own apps for monitoring PA, yet 16.12% (720/4467) report using apps. Fewer adolescents (782/4413; 17.72%) own HRM/SW and 9.25% (408/4413) use HRM/SW. In this study, users of HRM/SW were 2.09 times (95% CI 1.64-2.67), whereas users of apps were 1.4 times (95% CI 1.15-1.74) more likely to meet PA recommendations of daily MVPA for at least 60 min compared with adolescents without HRM/SW or without apps. Conclusions To our knowledge, this is the first study that describes the situation in Finland with adolescents using PA trackers and its association with PA levels. Implications of the use of apps and HRM/SW by adolescents are discussed. PMID:28473304

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR.

PubMed

Wang, Weilan; Zijlstra, Ruurd T; Gänzle, Michael G

2017-05-15

Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 10 5 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r 2  = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis.

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS.

PubMed

Botezatu, Irina V; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

2011-12-15

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the "closed tube" format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the "open tube" format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5-SYBR Green I and Bio-Rad CFX96-EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a "lower level" into the range of those suitable for mutation scanning. Copyright © 2011 Elsevier Inc. All rights reserved.

HIV Diversity as a Biomarker for HIV Incidence Estimation: Including a High-Resolution Melting Diversity Assay in a Multiassay Algorithm

PubMed Central

Cousins, Matthew M.; Konikoff, Jacob; Laeyendecker, Oliver; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Koblin, Beryl A.; Wheeler, Darrell; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Brookmeyer, Ron

2014-01-01

Multiassay algorithms (MAAs) can be used to estimate cross-sectional HIV incidence. We previously identified a robust MAA that includes the BED capture enzyme immunoassay (BED-CEIA), the Bio-Rad Avidity assay, viral load, and CD4 cell count. In this report, we evaluated MAAs that include a high-resolution melting (HRM) diversity assay that does not require sequencing. HRM scores were determined for eight regions of the HIV genome (2 in gag, 1 in pol, and 5 in env). The MAAs that were evaluated included the BED-CEIA, the Bio-Rad Avidity assay, viral load, and the HRM diversity assay, using HRM scores from different regions and a range of region-specific HRM diversity assay cutoffs. The performance characteristics based on the proportion of samples that were classified as MAA positive by duration of infection were determined for each MAA, including the mean window period. The cross-sectional incidence estimates obtained using optimized MAAs were compared to longitudinal incidence estimates for three cohorts in the United States. The performance of the HRM-based MAA was nearly identical to that of the MAA that included CD4 cell count. The HRM-based MAA had a mean window period of 154 days and provided cross-sectional incidence estimates that were similar to those based on cohort follow-up. HIV diversity is a useful biomarker for estimating HIV incidence. MAAs that include the HRM diversity assay can provide accurate HIV incidence estimates using stored blood plasma or serum samples without a requirement for CD4 cell count data. PMID:24153134

Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance.

PubMed

Wahyuningsih, Hesty; K Cayami, Ferdy; Bahrudin, Udin; A Sobirin, Mochamad; Ep Mundhofir, Farmaditya; Mh Faradz, Sultana; Hisatome, Ichiro

2017-03-01

High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1 .

High resolution melting (HRM) analysis as a new tool for rapid identification of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum.

PubMed

Ren, Xingxing; Fu, Ying; Xu, Chenggang; Feng, Zhou; Li, Miao; Zhang, Lina; Zhang, Jianmin; Liao, Ming

2017-05-01

Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum represent the most common causative agents of chicken salmonellosis, which result in high mortality and morbidity throughout the world. It is difficult and laborious to discriminate these diseases based on biochemical or phenotypic methods. Herein, we report the development of a single nucleotide polymorphism (SNP) PCR-high resolution melt (PCR-HRM) assay for the detection and discrimination of both S. Pullorum and S. Gallinarun. The gene rfbS, which encodes a factor involved in the biosynthesis of ADP paratose in serogroup D of Salmonella, has been identified as a robust genetic marker for the identification of S. Pullorum and S. Gallinarun based on polymorphisms at positions 237 and 598. Therefore, PCR-HRM analyses were used to characterize this gene. A total of 15 reference and 33 clinical isolates of Salmonella and related Gram-negative bacteria were detected using 2 sets of primers. Our PCR-HRM assay could distinguish S. Pullorum from S. Gallinarun and other strains using the primer pair SP-237F/237R. Similarly, S. Gallinarun could be distinguished from S. Pullorum and other strains using primer set SG-598F/598R. These 2 assays showed high specificity (100%) for both S. Pullorum and S. Gallinarun; the sensitivity of these 2 assays was at least 100-fold greater than that of the allele-specific PCR assay. This present study demonstrated that HRM analysis represents a potent, simple, and economic tool for the rapid, specific, and sensitive detection of S. Pullorum and S. Gallinarun. Our approach also may aid efforts for purification of Avian Salmonella disease. © 2016 Poultry Science Association Inc.

Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes.

PubMed

White, Helen E; Hall, Victoria J; Cross, Nicholas C P

2007-11-01

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are 2 distinct neurodevelopmental disorders caused primarily by deficiency of specific parental contributions at an imprinted domain within the chromosomal region 15q11.2-13. Lack of paternal contribution results in PWS either by paternal deletion (approximately 70%) or maternal uniparental disomy (UPD) (approximately 25%). Most cases of AS result from the lack of a maternal contribution from this same region, by maternal deletion (70%) or paternal UPD (approximately 5%). Analysis of allelic methylation differences at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus differentiates the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS. Methylation-sensitive high-resolution melting-curve analysis (MS-HRM) using the DNA binding dye EvaGreen was used to analyze methylation differences at the SNRPN locus in anonymized DNA samples from individuals with PWS (n = 39) or AS (n = 31) and from healthy control individuals (n = 95). Results from the MS-HRM assay were compared to those obtained by use of a methylation-specific PCR (MSP) protocol that is used commonly in diagnostic practice. With the MS-HRM assay 97.6% of samples were unambiguously assigned to the 3 diagnostic categories (AS, PWS, normal) by use of automated calling with an 80% confidence percentage threshold, and the failure rate was 0.6%. One PWS sample showed a discordant result for the MS-HRM assay compared to MSP data. MS-HRM is a simple, rapid, and robust method for screening methylation differences at the SNRPN locus and could be used as a diagnostic screen for PWS and AS.

Implementing Human Resources Management (HRM) within Dutch VET Institutions: Examining the Fostering and Hindering Factors

ERIC Educational Resources Information Center

Runhaar, Piety; Sanders, Karin

2013-01-01

Vocational Education and Training (VET) Institutions face serious challenges, like the implementation of competence-based education and upcoming teacher shortages, which urge them to implement Human Resources Management policy and practices (HRM). The implementation of HRM, however, often stagnates. This paper describes a qualitative study--in…

Changes in HRM in Europe: A Longitudinal Comparative Study among 18 European Countries

ERIC Educational Resources Information Center

Nikandrou, Irene; Apospori, Eleni; Papalexandris, Nancy

2005-01-01

Purpose: To examine HRM strategies and practices and HRM position within organizations in various cultural, economic and sociopolitical contexts from a longitudinal perspective. Design/methodology/approach: The study uses the 1995 and 1999 Cranet data in a longitudinal methodological framework to explore the changes and trends in 18 European…

One-step simultaneous detection of Ureaplasma parvum and genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology.

PubMed

Payne, M S; Furfaro, L L; Tucker, R; Tan, L Y; Mokany, E

2017-08-01

Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10 -5  ng DNA (SV3) and 0.4 × 10 -6  ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. © 2017 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

The use of colonic and anorectal high-resolution manometry and its place in clinical work and in research.

PubMed

Dinning, P G; Carrington, E V; Scott, S M

2015-12-01

In the esophagus, high-resolution manometry (HRM) has become a standard diagnostic tool in the investigation of suspected motility disorders. However, at the opposite end of the digestive tract (i.e., the colon and anorectum), the use of HRM still remains in its infancy, with relatively few published studies in the scientific literature. Further, the clinical utility of those studies that have been performed is largely undetermined. This review assesses all of the HRM studies published to date from both the colon and anorectum, explores the catheter types used, and attempts to determine the worth of HRM over traditional 'low-resolution' recordings from the same regions. Ultimately, this review addresses whether HRM currently provides information that will benefit patient diagnosis and treatment. © 2015 John Wiley & Sons Ltd.

Monochromatic X-ray-induced thermal effect on four-reflection “nested” meV-monochromators: dynamical diffraction theory and finite-element analysis

NASA Astrophysics Data System (ADS)

Hu, Ling-Fei; Gao, Li-Dan; Li, Zhen-Jie; Wang, Shan-Feng; Sheng, Wei-Fan; Liu, Peng; Xu, Wei

2015-09-01

The high energy resolution monochromator (HRM) is widely used in inelastic scattering programs to detect phonons with energy resolution, down to the meV level. Although the large amount of heat from insertion devices can be reduced by a high heat-load monochromator, the unbalanced heat load on the inner pair of crystals in a nested HRM can affect its overall performance. Here, a theoretical analysis of the unbalanced heat load using dynamical diffraction theory and finite element analysis is presented. By utilizing the ray-tracing method, the performance of different HRM nesting configurations is simulated. It is suggested that the heat balance ratio, energy resolution, and overall spectral transmission efficiency are the figures of merit for evaluating the performance of nested HRMs. Although the present study is mainly focused on nested HRMs working at 57Fe nuclear resonant energy at 14.4 keV, it is feasible to extend this to other nested HRMs working at different energies.

Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

PubMed

Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson

2013-12-15

Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia.

PubMed

Osathanunkul, Maslin; Dheeranupattana, Srisulak; Rotarayanont, Siriphron; Sookkhee, Siriwoot; Osathanunkul, Khukrit; Madesis, Panagiotis

2017-12-02

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.

Coronary Plaque Morphology and the Anti-Inflammatory Impact of Atorvastatin: A Multicenter 18F-Fluorodeoxyglucose Positron Emission Tomographic/Computed Tomographic Study.

PubMed

Singh, Parmanand; Emami, Hamed; Subramanian, Sharath; Maurovich-Horvat, Pal; Marincheva-Savcheva, Gergana; Medina, Hector M; Abdelbaky, Amr; Alon, Achilles; Shankar, Sudha S; Rudd, James H F; Fayad, Zahi A; Hoffmann, Udo; Tawakol, Ahmed

2016-12-01

Nonobstructive coronary plaques manifesting high-risk morphology (HRM) associate with an increased risk of adverse clinical cardiovascular events. We sought to test the hypothesis that statins have a greater anti-inflammatory effect within coronary plaques containing HRM. In this prospective multicenter study, 55 subjects with or at high risk for atherosclerosis underwent 18 F-fluorodeoxyglucose positron emission tomographic/computed tomographic imaging at baseline and after 12 weeks of treatment with atorvastatin. Coronary arterial inflammation ( 18 F-fluorodeoxyglucose uptake, expressed as target-to-background ratio) was assessed in the left main coronary artery (LMCA). While blinded to the PET findings, contrast-enhanced computed tomographic angiography was performed to characterize the presence of HRM (defined as noncalcified or partially calcified plaques) in the LMCA. Arterial inflammation (target-to-background ratio) was higher in LMCA segments with HRM than those without HRM (mean±SEM: 1.95±0.43 versus 1.67±0.32 for LMCA with versus without HRM, respectively; P=0.04). Moreover, atorvastatin treatment for 12 weeks reduced target-to-background ratio more in LMCA segments with HRM than those without HRM (12 week-baseline Δtarget-to-background ratio [95% confidence interval]: -0.18 [-0.35 to -0.004] versus 0.09 [-0.06 to 0.26]; P=0.02). Furthermore, this relationship between coronary plaque morphology and change in LMCA inflammatory activity remained significant after adjusting for baseline low-density lipoprotein and statin dose (β=-0.27; P=0.038). In this first study to evaluate the impact of statins on coronary inflammation, we observed that the anti-inflammatory impact of statins is substantially greater within coronary plaques that contain HRM features. These findings suggest an additional mechanism by which statins disproportionately benefit individuals with more advanced atherosclerotic disease. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00703261. © 2016 The Authors.

Human Resource Management and Human Resource Development: Evolution and Contributions

ERIC Educational Resources Information Center

Richman, Nicole

2015-01-01

Research agrees that a high performance organization (HPO) cannot exist without an elevated value placed on human resource management (HRM) and human resource development (HRD). However, a complementary pairing of HRM and HRD has not always existed. The evolution of HRD from its roots in human knowledge transference to HRM and present day HRD…

Beyond the Paycheck: A Human Resources Management Guide for Leaders of Small Youth-Serving Organizations

ERIC Educational Resources Information Center

Casey, Thomas P.

2009-01-01

This guide aims to help organization leaders develop the tools and knowledge they need to create and use sound human resources management (HRM) systems and practices that support program success and sustainability. It identifies key components of HRM systems and discusses important considerations in designing HRM policies, procedures, and…

The Educational Needs of and the Future Labor Market Demand for Human Resource Managers

ERIC Educational Resources Information Center

Prock, Dan; Henson, Bob

1977-01-01

Reports and discusses results of a national survey of professionals from all fields of human resource management (HRM) and college recruiters on the content of HRM education, competency requirements of certain specialists, and the labor market for personnel administration and industrial relations (PAIR/HRM) professionals in the next three years.…

Highly Relevant Mentoring (HRM) as a Faculty Development Model for Web-Based Instruction

ERIC Educational Resources Information Center

Carter, Lorraine; Salyers, Vincent; Page, Aroha; Williams, Lynda; Albl, Liz; Hofsink, Clarence

2012-01-01

This paper describes a faculty development model called the highly relevant mentoring (HRM) model; the model includes a framework as well as some practical strategies for meeting the professional development needs of faculty who teach web-based courses. The paper further emphasizes the need for faculty and administrative buy-in for HRM and…

Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica.

PubMed

Rojas, Alicia; Rojas, Diana; Montenegro, Víctor M; Baneth, Gad

2015-03-23

Canine filarioids are important nematodes transmitted to dogs by arthropods. Diagnosis of canine filariosis is accomplished by the microscopic identification of microfilariae, serology or PCR for filarial-DNA. The aim of this study was to evaluate a molecular assay for the detection of canine filariae in dog blood, to compare its performance to other diagnostic techniques, and to determine the relationship between microfilarial concentration and infection with other vector-borne pathogens. Blood samples from 146 dogs from Costa Rica were subjected to the detection of canine filarioids by four different methods: the microhematocrit tube test (MCT), Knott's modified test, serology and a high resolution melt and quantitative real-time PCR (HRM-qPCR). Co-infection with other vector-borne pathogens was also evaluated. Fifteen percent of the dogs were positive to Dirofilaria immitis by at least one of the methods. The HRM-qPCR produced distinctive melting plots for the different filarial worms and revealed that 11.6% of dogs were infected with Acanthocheilonema reconditum. The latter assay had a limit of detection of 2.4x10⁻⁴ mf/μl and detected infections with lower microfilarial concentrations in comparison to the microscopic techniques and the serological assay. The MCT and Knott's test only detected dogs with D. immitis microfilaremias above 0.7 mf/μl. Nevertheless, there was a strong correlation between the microfilarial concentration obtained by the Knott's modified test and the HRM-qPCR (r = 0.906, p < 0.0001). Interestingly, one dog was found infected with Cercopithifilaria bainae infection. Moreover, no association was found between microfilaremia and co-infection and there was no significant difference in microfilarial concentration between dogs infected only with D. immitis and dogs co-infected with Ehrlichia canis, Anaplasma platys or Babesia vogeli. This is the first report of A. reconditum and C. bainae in Costa Rica and Central America. Among the evaluated diagnostic techniques, the HRM-qPCR showed the most sensitive and reliable performance in the detection of blood filaroids in comparison to the Knott's modified test, the MCT test and a serological assay.

Global model for the lithospheric strength and effective elastic thickness

NASA Astrophysics Data System (ADS)

Tesauro, Magdala; Kaban, Mikhail K.; Cloetingh, Sierd A. P. L.

2013-08-01

Global distribution of the strength and effective elastic thickness (Te) of the lithosphere are estimated using physical parameters from recent crustal and lithospheric models. For the Te estimation we apply a new approach, which provides a possibility to take into account variations of Young modulus (E) within the lithosphere. In view of the large uncertainties affecting strength estimates, we evaluate global strength and Te distributions for possible end-member 'hard' (HRM) and a 'soft' (SRM) rheology models of the continental crust. Temperature within the lithosphere has been estimated using a recent tomography model of Ritsema et al. (2011), which has much higher horizontal resolution than previous global models. Most of the strength is localized in the crust for the HRM and in the mantle for the SRM. These results contribute to the long debates on applicability of the "crème brulée" or "jelly-sandwich" model for the lithosphere structure. Changing from the SRM to HRM turns most of the continental areas from the totally decoupled mode to the fully coupled mode of the lithospheric layers. However, in the areas characterized by a high thermal regime and thick crust, the layers remain decoupled even for the HRM. At the same time, for the inner part of the cratons the lithospheric layers are coupled in both models. Therefore, rheological variations lead to large changes in the integrated strength and Te distribution in the regions characterized by intermediate thermal conditions. In these areas temperature uncertainties have a greater effect, since this parameter principally determines rheological behavior. Comparison of the Te estimates for both models with those determined from the flexural loading and spectral analysis shows that the 'hard' rheology is likely applicable for cratonic areas, whereas the 'soft' rheology is more representative for young orogens.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

PubMed

Araujo, Sergio; Goulart, Luiz Ricardo; Truman, Richard W; Goulart, Isabela Maria B; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B; Rosa, Patricia S; Scollard, David M; Williams, Diana L

2017-06-01

Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

Expiratory Muscle Strength Training Evaluated With Simultaneous High Resolution Manometry and Electromyography

PubMed Central

Hutcheson, Katherine A.; Hammer, Michael J.; Rosen, Sarah P.; Jones, Corinne A.; McCulloch, Timothy M.

2017-01-01

Objective To examine feasibility of a simultaneous high-resolution pharyngeal manometry (HRM) and electromyography (EMG) experimental paradigm to detect swallowing-related patterns of palatal, laryngeal, and pharyngeal muscle activity during expiratory training. Study Design Technical report. Methods Simultaneous HRM, surface submental, and intramuscular EMG were acquired in two healthy participants during five tasks: 10-cc water swallow, maximum expiratory pressure (MEP) testing, and expiratory muscle strength training (EMST) at three pressure levels (sham, 50%, and 75% MEP). Results Experimental conditions were feasible. Velopharyngeal closing pressure, palate EMG activity, and pharyngeal EMG activity increased as expiratory load increased. In contrast, thyroarytenoid EMG activity was low during the expiratory task, consistent with glottic opening during exhalation. Submental EMG patterns were more variable during expiratory tasks. Intraluminal air pressures recorded with HRM were correlated with measured expiratory pressures and target valve-opening pressures of the EMST device. Conclusion Results suggest that a simultaneous HRM/EMG/EMST paradigm may be used to detect previously unquantified swallowing-related muscle activity during EMST, particularly in the palate and pharynx. Our approach and initial findings will be helpful to guide future hypothesis-driven studies and may enable investigators to evaluate other muscle groups active during these tasks. Defining mechanisms of action is a critical next step toward refining therapeutic algorithms using EMST and other targeted treatments for populations with dysphagia and airway disorders. PMID:28083946

Ultrasensitive Detection of Drug-Resistant Pandemic 2009 (H1N1) Influenza A Virus by Rare-Variant-Sensitive High-Resolution Melting-Curve Analysis▿‡

PubMed Central

Chen, Neng; Pinsky, Benjamin A.; Lee, Betty P.; Lin, Min; Schrijver, Iris

2011-01-01

Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality. PMID:21543559

A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species.

PubMed

Kirkpatrick, Naomi C; Blacker, Hayley P; Woods, Wayne G; Gasser, Robin B; Noormohammadi, Amir H

2009-02-01

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as "variation" to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.

Bluetooth Heart Rate Monitors For Spaceflight

NASA Technical Reports Server (NTRS)

Buxton, R. E.; West, M. R.; Kalogera, K. L.; Hanson, A. M.

2016-01-01

Heart rate monitoring is required for crewmembers during exercise aboard the International Space Station (ISS) and will be for future exploration missions. The cardiovascular system must be sufficiently stressed throughout a mission to maintain the ability to perform nominal and contingency/emergency tasks. High quality heart rate data are required to accurately determine the intensity of exercise performed by the crewmembers and show maintenance of VO2max. The quality of the data collected on ISS is subject to multiple limitations and is insufficient to meet current requirements. PURPOSE: To evaluate the performance of commercially available Bluetooth heart rate monitors (BT_HRM) and their ability to provide high quality heart rate data to monitor crew health aboard the ISS and during future exploration missions. METHODS: Nineteen subjects completed 30 data collection sessions of various intensities on the treadmill and/or cycle. Subjects wore several BT_HRM technologies for each testing session. One electrode-based chest strap (CS) was worn, while one or more optical sensors (OS) were worn. Subjects were instrumented with a 12-lead ECG to compare the heart rate data from the Bluetooth sensors. Each BT_HRM data set was time matched to the ECG data and a +/-5bpm threshold was applied to the difference between the 2 data sets. Percent error was calculated based on the number of data points outside the threshold and the total number of data points. RESULTS: The electrode-based chest straps performed better than the optical sensors. The best performing CS was CS1 (1.6% error), followed by CS4 (3.3% error), CS3 (6.4% error), and CS2 (9.2% error). The OS resulted in 10.4% error for OS1 and 14.9% error for OS2. CONCLUSIONS: The highest quality data came from CS1, but unfortunately it has been discontinued by the manufacturer. The optical sensors have not been ruled out for use, but more investigation is needed to determine how to obtain the best quality data. CS2 will be used in an ISS Bluetooth validation study, because it simultaneously transmits magnetic pulse that is integrated with existing exercise hardware on ISS. The simultaneous data streams allow for beat-to-beat comparison between the current ISS standard and CS2. Upon Bluetooth validation aboard ISS, the research team will down select a new BT_HRM for operational use.

High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

PubMed

Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

2014-01-01

A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanisms of repetitive retrograde contractions in response to sustained esophageal distension: a study evaluating patients with postfundoplication dysphagia.

PubMed

Carlson, Dustin A; Kahrilas, Peter J; Ritter, Katherine; Lin, Zhiyue; Pandolfino, John E

2018-03-01

Repetitive retrograde contractions (RRCs) in response to sustained esophageal distension are a distinct contractility pattern observed with functional luminal imaging probe (FLIP) panometry that are common in type III (spastic) achalasia. RRCs are hypothesized to be indicative of either impaired inhibitory innervation or esophageal outflow obstruction. We aimed to apply FLIP panometry to patients with postfundoplication dysphagia (a model of esophageal obstruction) to explore mechanisms behind RRCs. Adult patients with dysphagia after Nissen fundoplication ( n = 32) or type III achalasia ( n = 25) were evaluated with high-resolution manometry (HRM) and upper endoscopy with FLIP. HRM studies were assessed for outflow obstruction and spastic features: premature contractility, hypercontractility, and impaired deglutitive inhibition during multiple-rapid swallows. FLIP studies were analyzed to determine the esophagogastric junction (EGJ)-distensibility index and contractility pattern, including RRCs. Barium esophagram was evaluated when available. RRCs were present in 8/32 (25%) fundoplication and 19/25 (76%) achalasia patients ( P < 0.001). EGJ outflow obstruction was detected in 21 (67%) fundoplication patients by HRM, FLIP, or esophagram [6 (29%) had RRCs]. On HRM, none of the fundoplication patients had premature contractility, whereas 3/4 with defective inhibition on multiple-rapid swallows and 2/4 with hypercontractility had RRCs. Regression analysis demonstrated HRM with spastic features, but not esophageal outflow obstruction, as a predictor for RRCs. RRCs in response to sustained esophageal distension appear to be a manifestation of spastic esophageal motility. Although future study to further clarify the significance of RRCs is needed, RRCs on FLIP panometry should prompt evaluation for a major motor disorder. NEW & NOTEWORTHY Repetitive retrograde contractions (RRCs) are a common response to sustained esophageal distension among spastic achalasia patients when evaluated with the functional luminal imaging probe. We evaluated patients with postfundoplication dysphagia, i.e., patients with suspected mechanical obstruction, and found that RRCs occasionally occurred among postfundoplication patients, but often in association with manometric features of esophageal neuromuscular imbalance. Thus, RRCs appear to be a manifestation of spastic esophageal dysmotility, likely from neural imbalance resulting in excess excitation.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis.

PubMed

Sulyok, Kinga M; Bekő, Katinka; Kreizinger, Zsuzsa; Wehmann, Enikő; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Szeredi, Levente; Jánosi, Szilárd; Nagy, Sára Ágnes; Gyuranecz, Miklós

2018-01-01

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 10 2 -10 5 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

PubMed

Daniels, Rachel; Hamilton, Elizabeth J; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F; Hartl, Daniel L; Volkman, Sarah K

2015-11-10

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs. This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

Esophageal manometry in gastroesophageal reflux disease.

PubMed

Mello, Michael; Gyawali, C Prakash

2014-03-01

High-resolution manometry (HRM) allows nuanced evaluation of esophageal motor function, and more accurate evaluation of lower esophageal sphincter (LES) function, in comparison with conventional manometry. Pathophysiologic correlates of gastroesophageal reflux disease (GERD) and esophageal peristaltic performance are well addressed by this technique. HRM may alter the surgical decision by assessment of esophageal peristaltic function and exclusion of esophageal outflow obstruction before antireflux surgery. Provocative testing during HRM may assess esophageal smooth muscle peristaltic reserve and help predict the likelihood of transit symptoms following antireflux surgery. HRM represents a continuously evolving new technology that compliments the evaluation and management of GERD. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of esophageal motor function in clinical practice.

PubMed

Gyawali, C P; Bredenoord, A J; Conklin, J L; Fox, M; Pandolfino, J E; Peters, J H; Roman, S; Staiano, A; Vaezi, M F

2013-02-01

Esophageal motor function is highly coordinated between central and enteric nervous systems and the esophageal musculature, which consists of proximal skeletal and distal smooth muscle in three functional regions, the upper and lower esophageal sphincters, and the esophageal body. While upper endoscopy is useful in evaluating for structural disorders of the esophagus, barium esophagography, radionuclide transit studies, and esophageal intraluminal impedance evaluate esophageal transit and partially assess motor function. However, esophageal manometry is the test of choice for the evaluation of esophageal motor function. In recent years, high-resolution manometry (HRM) has streamlined the process of acquisition and display of esophageal pressure data, while uncovering hitherto unrecognized esophageal physiologic mechanisms and pathophysiologic patterns. New algorithms have been devised for analysis and reporting of esophageal pressure topography from HRM. The clinical value of HRM extends to the pediatric population, and complements preoperative evaluation prior to foregut surgery. Provocative maneuvers during HRM may add to the assessment of esophageal motor function. The addition of impedance to HRM provides bolus transit data, but impact on clinical management remains unclear. Emerging techniques such as 3-D HRM and impedance planimetry show promise in the assessment of esophageal sphincter function and esophageal biomechanics. © 2013 Blackwell Publishing Ltd.

Domestic Banks in Bangladesh Could Ensure Efficiency by Improving Human Resource Management Practices

PubMed Central

Muhammad Masum, Abdul Kadar; Azad, Md. Abul Kalam; Hoque, Kazi Enamul; Beh, Loo-See

2015-01-01

The paper aims to examine the influence of human resource management (HRM) practices on bank efficiency using Malmquist index of total factor productivity. The model comprises HRM index that represents the quality of HRM practices. The results are decomposed into three efficiency scores, namely, technical efficiency, pure efficiency, and scale efficiency. In this study, panel data for 44 banks in Bangladesh are used for the period 2008-2013. This paper reveals that foreign banks are ahead in converting the influence of HRM practices into efficiency scores (0.946>0.833). On the other hand, domestic banks performed better than foreign banks in terms of pure efficiency and scale efficiency. But, in terms of technical efficiency, the domestic banks are regressed by 6.7% annually whereas foreign banks are progressed with a yearly value of 5.8%. The results are robust, because the Mann-Whitney test and Kruskall-Wallis test (non-parametric tests) also confirm the same results. This study emphasizes HRM practices in the banking industry to ensure efficiency in the long-term scenario. Domestic banks are suggested to ensure continuous development in HRM practices in order to compete with foreign banks. PMID:26221727

Domestic Banks in Bangladesh Could Ensure Efficiency by Improving Human Resource Management Practices.

PubMed

Masum, Abdul Kadar Muhammad; Azad, Md Abul Kalam; Hoque, Kazi Enamul; Beh, Loo-See

2015-01-01

The paper aims to examine the influence of human resource management (HRM) practices on bank efficiency using Malmquist index of total factor productivity. The model comprises HRM index that represents the quality of HRM practices. The results are decomposed into three efficiency scores, namely, technical efficiency, pure efficiency, and scale efficiency. In this study, panel data for 44 banks in Bangladesh are used for the period 2008-2013. This paper reveals that foreign banks are ahead in converting the influence of HRM practices into efficiency scores (0.946>0.833). On the other hand, domestic banks performed better than foreign banks in terms of pure efficiency and scale efficiency. But, in terms of technical efficiency, the domestic banks are regressed by 6.7% annually whereas foreign banks are progressed with a yearly value of 5.8%. The results are robust, because the Mann-Whitney test and Kruskall-Wallis test (non-parametric tests) also confirm the same results. This study emphasizes HRM practices in the banking industry to ensure efficiency in the long-term scenario. Domestic banks are suggested to ensure continuous development in HRM practices in order to compete with foreign banks.

Correlation of High-Resolution Manometric Findings with Symptoms of Dysphagia and Endoscopic Features in Adults with Eosinophilic Esophagitis.

PubMed

von Arnim, Ulrike; Kandulski, Arne; Weigt, Jochen; Malfertheiner, Peter

2017-01-01

Eosinophilic esophagitis (EoE) presents with dysphagia, but data about motility patterns using high-resolution manometry (HRM) are rare. We aimed at evaluating esophageal motility patterns in EoE and their correlation to endoscopic and dysphagia scores. Twenty-six EoE patients and 23 controls were included after 4 weeks of treatment with proton pump inhibitors. Dysphagia and endoscopic scores were evaluated before performing HRM. EoE patients were classified to have fibrostenotic (FS) or inflammatory (IF) type. HRM analysis was performed according to the Chicago classification (CC) system. According to the CC, the HRM findings in EoE and controls were normal in 11 (42%) and 20 (88.5%), p < 0.0001. Weak and failed peristaltic integrity was only seen in EoE patients (failed 1/2.7%, weak 7/26.9%, p = 0.004). Of the EoE patients, 17 had IF and 9 presented with FS type. HRM parameters showed no differences according to the EoE subtype. The endoscopic score in the FS subtype was significantly higher than in EoE with IF subtype (5.33 vs. 3.58, p = 0.001). No significant difference was seen in dysphagia scores in EoE subtypes. HRM findings in EoE are often diagnostic, but they are non-specific and do not correlate with the severity of dysphagia or endoscopic appearance. The clinical impact of HRM in EoE needs further evaluation. © 2017 S. Karger AG, Basel.

Classification of esophageal motor findings in gastro-esophageal reflux disease: Conclusions from an international consensus group.

PubMed

Gyawali, C P; Roman, S; Bredenoord, A J; Fox, M; Keller, J; Pandolfino, J E; Sifrim, D; Tatum, R; Yadlapati, R; Savarino, E

2017-12-01

High-resolution manometry (HRM) has resulted in new revelations regarding the pathophysiology of gastro-esophageal reflux disease (GERD). The impact of new HRM motor paradigms on reflux burden needs further definition, leading to a modern approach to motor testing in GERD. Focused literature searches were conducted, evaluating pathophysiology of GERD with emphasis on HRM. The results were discussed with an international group of experts to develop a consensus on the role of HRM in GERD. A proposed classification system for esophageal motor abnormalities associated with GERD was generated. Physiologic gastro-esophageal reflux is inherent in all humans, resulting from transient lower esophageal sphincter (LES) relaxations that allow venting of gastric air in the form of a belch. In pathological gastro-esophageal reflux, transient LES relaxations are accompanied by reflux of gastric contents. Structural disruption of the esophagogastric junction (EGJ) barrier, and incomplete clearance of the refluxate can contribute to abnormally high esophageal reflux burden that defines GERD. Esophageal HRM localizes the LES for pH and pH-impedance probe placement, and assesses esophageal body peristaltic performance prior to invasive antireflux therapies and antireflux surgery. Furthermore, HRM can assess EGJ and esophageal body mechanisms contributing to reflux, and exclude conditions that mimic GERD. Structural and motor EGJ and esophageal processes contribute to the pathophysiology of GERD. A classification scheme is proposed incorporating EGJ and esophageal motor findings, and contraction reserve on provocative tests during HRM. © 2017 John Wiley & Sons Ltd.

The Hierarchical Rater Model for Rated Test Items and Its Application to Large-Scale Educational Assessment Data.

ERIC Educational Resources Information Center

Patz, Richard J.; Junker, Brian W.; Johnson, Matthew S.; Mariano, Louis T.

2002-01-01

Discusses the hierarchical rater model (HRM) of R. Patz (1996) and shows how it can be used to scale examinees and items, model aspects of consensus among raters, and model individual rater severity and consistency effects. Also shows how the HRM fits into the generalizability theory framework. Compares the HRM to the conventional item response…

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

PubMed Central

Thomas, Holly R.; Percival, Stefanie M.; Yoder, Bradley K.; Parant, John M.

2014-01-01

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism. PMID:25503746

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

PubMed Central

Goulart, Luiz Ricardo; Truman, Richard W.; Goulart, Isabela Maria B.; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B.; Rosa, Patricia S.; Scollard, David M.; Williams, Diana L.

2017-01-01

Background Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. Methodology/Principal findings The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite’s stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite’s stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. Conclusion/Significance Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy. PMID:28570560

Calculation of upper esophageal sphincter restitution time from high resolution manometry data using machine learning.

PubMed

Jungheim, Michael; Busche, Andre; Miller, Simone; Schilling, Nicolas; Schmidt-Thieme, Lars; Ptok, Martin

2016-10-15

After swallowing, the upper esophageal sphincter (UES) needs a certain amount of time to return from maximum pressure to the resting condition. Disturbances of sphincter function not only during the swallowing process but also in this phase of pressure restitution may lead to globus sensation or dysphagia. Since UES pressures do not decrease in a linear or asymptotic manner, it is difficult to determine the exact time when the resting pressure is reached, even when using high resolution manometry (HRM). To overcome this problem a Machine Learning model was established to objectively determine the UES restitution time (RT) and moreover to collect physiological data on sphincter function after swallowing. HRM-data of 15 healthy participants performing 10 swallows each were included. After manual annotation of the RT interval by two swallowing experts, data were transferred to the Machine Learning model, which applied a sequence labeling modeling approach based on logistic regression to learn and objectivize the characteristics of all swallows. Individually computed RT values were then compared with the annotated values. Estimates of the RT were generated by the Machine Learning model for all 150 swallows. When annotated by swallowing experts mean RT of 11.16s±5.7 (SD) and 10.04s±5.74 were determined respectively, compared to model-generated values from 8.91s±3.71 to 10.87s±4.68 depending on model selection. The correlation score for the annotated RT of both examiners was 0.76 and 0.63 to 0.68 for comparison of model predicted values. Restitution time represents an important physiologic swallowing parameter not previously considered in HRM-studies of the UES, especially since disturbances of UES restitution may increase the risk of aspiration. The data presented here show that it takes approximately 9 to 11s for the UES to come to rest after swallowing. Based on maximal RT values, we demonstrate that an interval of 25-30s in between swallows is necessary until the next swallow is initiated. This should be considered in any further HRM-studies designed to evaluate the characteristics of individual swallows. The calculation model enables a quick and reproducible determination of the time it takes for the UES to come to rest after swallowing (RT). The results of the calculation are partially independent of the input of the investigator. Adding more swallows and integrating additional parameters will improve the Machine Leaning model in the future. By applying similar models to other swallowing parameters of the pharynx and UES, such as the relaxation time of the UES or the activity time during swallowing, a complete automatic evaluation of HRM-data of a swallow should be possible. Copyright © 2016 Elsevier Inc. All rights reserved.

Implementation of High-resolution Manometry in the Clinical Practice of Speech Language Pathology

PubMed Central

Thibeault, Susan; McCulloch, Timothy M.

2014-01-01

Visual imaging modalities, videofluoroscopic swallow study (VFSS) and fiberoptic endoscopic evaluation of swallow, for assessment of oropharyngeal dysphagia have been part of the speech language pathologist’s (SLPs) armamentarium for the diagnosis and treatment of dysphagia for decades. Recently, the addition of high-resolution manometry (HRM) has enabled the SLP to evaluate pharyngeal pressures and upper esophageal sphincter relaxation. Taken together, the use of visual imaging modalities with HRM can improve interpretation of swallowing physiology and facilitate more effective treatment planning. The goal of this article is to describe a clinical paradigm using HRM as an adjunct to VFSS, by the SLP, in the assessment of complex dysphagia. Moreover, in three cases described, the value of manometric measurements in elucidating swallowing imaging studies and documenting physiologic change in response to treatment is highlighted. As technology in this area is evolving, so will the clinical use of HRM by the SLP. Limitations of current HRM systems and applications are discussed. PMID:24233810

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

PubMed Central

Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

2013-01-01

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

Cysteamine treatment ameliorates alterations in GAD67 expression and spatial memory in heterozygous reeler mice

PubMed Central

Kutiyanawalla, Ammar; Promsote, Wanwisa; Terry, Alvin; Pillai, Anilkumar

2011-01-01

Brain derived neurotrophic factor (BDNF) signaling through its receptor, TrkB is known to regulate GABAergic function and glutamic acid decarboxylase (GAD) 67 expression in neurons. Alterations in BDNF signaling have been implicated in the pathophysiology of schizophrenia and as a result, they are a potential therapeutic target. Interestingly, heterozygous reeler mice (HRM) have decreased GAD67 expression in the frontal cortex and hippocampus and they exhibit many behavioral and neurochemical abnormalities similar to schizophrenia. In the present study, we evaluated the potential of cysteamine, a neuroprotective compound to improve the deficits in GAD67 expression and cognitive function in HRM. We found that cysteamine administration (150 mg/kg/day, through drinking water) for 30 days significantly ameliorated the decreases in GAD67, mature BDNF and full-length TrkB protein levels found in frontal cortex and hippocampus of HRM. A significant attenuation of the increased levels of truncated BDNF in frontal cortex and hippocampus, as well as truncated TrkB in frontal cortex of HRM was also observed following cysteamine treatment. In behavioral studies, HRM were impaired in a Y-maze spatial recognition memory task, but not in a spontaneous alternation task or a sensorimotor, prepulse inhibition (PPI) procedure. Cysteamine improved Y-maze spatial recognition in HRM to the level of wide-type controls and it improved PPI in both wild-type and HRM. Finally, mice deficient in TrkB, showed a reduced response to cysteamine in GAD67 expression suggesting that TrkB signaling plays an important role in GAD67 regulation by cysteamine. PMID:21777509

Measurement of dynamic urethral pressures with a high-resolution manometry system in continent and incontinent women.

PubMed

Kirby, Anna C; Tan-Kim, Jasmine; Nager, Charles W

2015-01-01

Female stress urinary incontinence is caused by urethral dysfunction during dynamic conditions, but current technology has limitations in measuring urethral pressures under these conditions. An 8-French high-resolution manometry (HRM) catheter currently in clinical use in gastroenterology may accurately measure urethral pressures under dynamic conditions because it has a 25-millisecond response rate and circumferential pressure sensors along the length of the catheter (ManoScan ESO; Given Imaging, Yoqneam, Israel). We evaluated the concordance, repeatability, and tolerability of this catheter. We measured resting, cough, and strain maximum urethral closure pressures (MUCPs) using HRM and measured resting MUCPs with water-perfusion side-hole catheter urethral pressure profilometry (UPP) in 37 continent and 28 stress-incontinent subjects. Maneuvers were repeated after moving the HRM catheter along the urethral length to evaluate whether results depend on catheter positioning. Visual analog pain scores evaluated the comfort of HRM compared to UPP. The correlation coefficient for resting MUCPs measured by HRM versus UPP was high (r = 0.79, P < 0.001). Repeatability after catheter repositioning was high for rest, cough, and strain with HRM: r = 0.92, 0.89, and 0.89. Mean MUCPs (rest, cough, and strain) were higher in continent than in incontinent subjects (all P < 0.001) and decreased more in incontinent subjects than in continent subjects during cough and strain maneuvers compared to rest. This preliminary study shows that HRM is concordant with standard technology, repeatable, and well tolerated in the urethra. Incontinent women have more impairment of their urethral closure pressures during cough and strain than continent women.

Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains

PubMed Central

Ghorashi, Seyed A.; Kanci, Anna; Noormohammadi, Amir H.

2015-01-01

Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10-4 ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population. PMID:25970590

High-resolution melting analysis of sequence variations in the cytidine deaminase gene (CDA) in patients with cancer treated with gemcitabine.

PubMed

Raynal, Caroline; Ciccolini, Joseph; Mercier, Cédric; Boyer, Jean-Christophe; Polge, Anne; Lallemant, Benjamin; Mouzat, Kévin; Lumbroso, Serge; Brouillet, Jean-Paul; Evrard, Alexandre

2010-02-01

Gemcitabine (2',2'-difluorodeoxycytidine) is a major antimetabolite cytotoxic drug with a wide spectrum of activity against solid tumors. Hepatic elimination of gemcitabine depends on a catabolic pathway through a deamination step driven by the enzyme cytidine deaminase (CDA). Severe hematologic toxicity to gemcitabine was reported in patients harboring genetic polymorphisms in CDA gene. High-resolution melting (HRM) analysis of polymerase chain reaction amplicon emerges today as a powerful technique for both genotyping and gene scanning strategies. In this study, 46 DNA samples from gemcitabine-treated patients were subjected to HRM analysis on a LightCycler 480 platform. Residual serum CDA activity was assayed as a surrogate marker for the overall functionality of this enzyme. Genotyping of three well-described single nucleotide polymorphisms in coding region (c.79A>C, c.208G>A and c.435C>T) was successfully achieved by HRM analysis of small polymerase chain reaction fragments, whereas unknown single nucleotide polymorphisms were searched by a gene scanning strategy with longer amplicons (up to 622 bp). The gene scanning strategy allowed us to find a new intronic mutation c.246+37G>A in a female patient displaying marked CDA deficiency and who had an extreme toxic reaction with a fatal outcome to gemcitabine treatment. Our work demonstrates that HRM-based methods, owing to their simplicity, reliability, and speed, are useful tools for diagnosis of CDA deficiency and could be of interest for personalized medicine.

First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis.

PubMed

Hosseini-Safa, Ahmad; Mohag Hegh, Mohammad Ali; Pestechian, Nader; Ganji, Maryam; Mohammadi, Rasoul; Mahmoudi Lamouki, Reza; Rostami-Nejad, Mohammad

2016-12-01

The present study was aimed to evaluate E. granulosus genotypes isolated from goats using HRM analysis in Isfahan province. Cystic echincoccosis, so-called hydatidosis, is widespread infection caused by the larval stage of Echinococcus granulosus . This is an important zoonotic disease worldwide, especially in the developing countries such as Iran. To date, molecular studies mainly based on the mitochondrial DNA sequences have identified distinct genotypes termed G1-G10 which can differ in some characteristics such as the growth and infectivity to different intermediate hosts or the survival rate in the definitive hosts that are important for the development of control strategies. From August to December 2014, 1341 goats were investigated and hydatid cysts were collected from the liver and lungs of 43 infected goats in Isfahan province abattoirs, Isfahan, Iran. Total genomic DNA was extracted from each sample, amplified for the presence of polymorphism of mitochondrial gene coding for cytochrome c oxidase subunit 1 (CO1), using high resolution melting curve (HRM) method. the results of HRM analysis using the sequence of CO1 gene for 43 Echinococcus granulosus isolates from goats showed 31, 2 and 10 isolates were identified as G1, G2, and G3 genotypes, respectively. G1 is the predominant genotype in the isolated goat samples in Isfahan province, and the presence of G2 strain was reported for the first time in goat in Iran.

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes.

PubMed

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-09-11

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica , about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N 50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both "Zheda 23" and "Zheda 83". Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.

Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

PubMed

Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

2015-11-01

Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

HRM and its effect on employee, organizational and financial outcomes in health care organizations

PubMed Central

2014-01-01

Background One of the main goals of Human Resource Management (HRM) is to increase the performance of organizations. However, few studies have explicitly addressed the multidimensional character of performance and linked HR practices to various outcome dimensions. This study therefore adds to the literature by relating HR practices to three outcome dimensions: financial, organizational and employee (HR) outcomes. Furthermore, we will analyze how HR practices influence these outcome dimensions, focusing on the mediating role of job satisfaction. Methods This study uses a unique dataset, based on the ‘ActiZ Benchmark in Healthcare’, a benchmark study conducted in Dutch home care, nursing care and care homes. Data from autumn 2010 to autumn 2011 were analyzed. In total, 162 organizations participated during this period (approximately 35% of all Dutch care organizations). Employee data were collected using a questionnaire (61,061 individuals, response rate 42%). Clients were surveyed using the Client Quality Index for long-term care, via stratified sampling. Financial outcomes were collected using annual reports. SEM analyses were conducted to test the hypotheses. Results It was found that HR practices are - directly or indirectly - linked to all three outcomes. The use of HR practices is related to improved financial outcomes (measure: net margin), organizational outcomes (measure: client satisfaction) and HR outcomes (measure: sickness absence). The impact of HR practices on HR outcomes and organizational outcomes proved substantially larger than their impact on financial outcomes. Furthermore, with respect to HR and organizational outcomes, the hypotheses concerning the full mediating effect of job satisfaction are confirmed. This is in line with the view that employee attitudes are an important element in the ‘black box’ between HRM and performance. Conclusion The results underscore the importance of HRM in the health care sector, especially for HR and organizational outcomes. Further analyses of HRM in the health care sector will prove to be a productive endeavor for both scholars and HR managers. PMID:24938460

Clinically led performance management in secondary healthcare: evaluating the attitudes of medical and non-clinical managers.

PubMed

Trebble, Timothy M; Paul, Maureen; Hockey, Peter M; Heyworth, Nicola; Humphrey, Rachael; Powell, Timothy; Clarke, Nicholas

2015-03-01

Improving the quality and activity of clinicians' practice improves patient care. Performance-related human resource management (HRM) is an established approach to improving individual practice but with limited use among clinicians. A framework for performance-related HRM was developed from successful practice in non-healthcare organisations centred on distributive leadership and locally provided, validated and interpreted performance measurement. This study evaluated the response of medical and non-clinical managers to its implementation into a large secondary healthcare organisation. A semistructured qualitative questionnaire was developed from themes identified during framework implementation and included attitudes to previous approaches to measuring doctors' performance, and the structure and response to implementation of the performance-related HRM framework. Responses were analysed through a process of data summarising and categorising. A total of 29, from an invited cohort of 31, medical and non-clinical managers from departmental to executive level were interviewed. Three themes were identified: (1) previous systems of managing clinical performance were considered to be ineffective due to insufficient empowerment of medical managers and poor quality of available performance data; (2) the implemented framework was considered to address these needs and was positively received by medical and non-clinical managers; (3) introduction of performance-related HRM required the involvement of the whole organisation to executive level and inclusion within organisational strategy, structure and training. This study suggests that a performance-related HRM framework may facilitate the management of clinical performance in secondary healthcare, but is dependent on the design and methods of application used. Such approaches contrast with those currently proposed for clinicians in secondary healthcare in the UK and suggest that alternative strategies should be considered. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Application of process mapping to understand integration of high risk medicine care bundles within community pharmacy practice.

PubMed

Weir, Natalie M; Newham, Rosemary; Corcoran, Emma D; Ali Atallah Al-Gethami, Ashwag; Mohammed Abd Alridha, Ali; Bowie, Paul; Watson, Anne; Bennie, Marion

2017-11-21

The Scottish Patient Safety Programme - Pharmacy in Primary Care collaborative is a quality improvement initiative adopting the Institute of Healthcare Improvement Breakthrough Series collaborative approach. The programme developed and piloted High Risk Medicine (HRM) Care Bundles (CB), focused on warfarin and non-steroidal anti-inflammatories (NSAIDs), within 27 community pharmacies over 4 NHS Regions. Each CB involves clinical assessment and patient education, although the CB content varies between regions. To support national implementation, this study aims to understand how the pilot pharmacies integrated the HRM CBs into routine practice to inform the development of a generic HRM CB process map. Regional process maps were developed in 4 pharmacies through simulation of the CB process, staff interviews and documentation of resources. Commonalities were collated to develop a process map for each HRM, which were used to explore variation at a national event. A single, generic process map was developed which underwent validation by case study testing. The findings allowed development of a generic process map applicable to warfarin and NSAID CB implementation. Five steps were identified as required for successful CB delivery: patient identification; clinical assessment; pharmacy CB prompt; CB delivery; and documentation. The generic HRM CB process map encompasses the staff and patients' journey and the CB's integration into routine community pharmacy practice. Pharmacist involvement was required only for clinical assessment, indicating suitability for whole-team involvement. Understanding CB integration into routine practice has positive implications for successful implementation. The generic process map can be used to develop targeted resources, and/or be disseminated to facilitate CB delivery and foster whole team involvement. Similar methods could be utilised within other settings, to allow those developing novel services to distil the key processes and consider their integration within routine workflows to effect maximal, efficient implementation and benefit to patient care. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

PubMed

Zianni, Michael R; Nikbakhtzadeh, Mahmood R; Jackson, Bryan T; Panescu, Jenny; Foster, Woodbridge A

2013-04-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

PubMed Central

Zianni, Michael R.; Nikbakhtzadeh, Mahmood R.; Jackson, Bryan T.; Panescu, Jenny; Foster, Woodbridge A.

2013-01-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software. PMID:23543777

Impact of human resource management practices on nursing home performance.

PubMed

Rondeau, K V; Wagar, T H

2001-08-01

Management scholars and practitioners alike have become increasingly interested in learning more about the ability of certain 'progressive' or 'high-performance' human resource management (HRM) practices to enhance organizational effectiveness. There is growing evidence to suggest that the contribution of various HRM practices to impact firm performance may be synergistic in effect yet contingent on a number of contextual factors, including workplace climate. A contingency theory perspective suggests that in order to be effective, HMR policies and practices must be consistent with other aspects of the organization, including its environment. This paper reports on empirical findings from research that examines the relationship between HRM practices, workplace climate and perceptions of organizational performance, in a large sample of Canadian nursing homes. Data from 283 nursing homes were collected by means of a mail survey that included questions on HRM practices, programmes, and policies, on human resource aspects of workplace climate, as well as a variety of indicators that include employee, customer/resident and facility measures of organizational performance. Results derived from ordered probit analysis suggest that nursing homes in our sample which had implemented more 'progressive' HRM practices and which reported a workplace climate that strongly values employee participation, empowerment and accountability tended to be perceived to generally perform better on a number of valued organizational outcomes. Nursing homes in our sample that performed best overall were found to be more likely to not only have implemented more of these HRM practices, but also to report having a workplace climate that reflects the seminal value that it places on its human resources. This finding is consistent with the conclusion that simply introducing HRM practices or programmes, in the absence of an appropriately supportive workplace climate, will be insufficient to attain optimal organizational performance.

Quantitative Analysis of Swallowing Function Between Dysphagia Patients and Healthy Subjects Using High-Resolution Manometry.

PubMed

Park, Chul-Hyun; Kim, Don-Kyu; Lee, Yong-Taek; Yi, Youbin; Lee, Jung-Sang; Kim, Kunwoo; Park, Jung Ho; Yoon, Kyung Jae

2017-10-01

To compare swallowing function between healthy subjects and patients with pharyngeal dysphagia using high resolution manometry (HRM) and to evaluate the usefulness of HRM for detecting pharyngeal dysphagia. Seventy-five patients with dysphagia and 28 healthy subjects were included in this study. Diagnosis of dysphagia was confirmed by a videofluoroscopy. HRM was performed to measure pressure and timing information at the velopharynx (VP), tongue base (TB), and upper esophageal sphincter (UES). HRM parameters were compared between dysphagia and healthy groups. Optimal threshold values of significant HRM parameters for dysphagia were determined. VP maximal pressure, TB maximal pressure, UES relaxation duration, and UES resting pressure were lower in the dysphagia group than those in healthy group. UES minimal pressure was higher in dysphagia group than in the healthy group. Receiver operating characteristic (ROC) analyses were conducted to validate optimal threshold values for significant HRM parameters to identify patients with pharyngeal dysphagia. With maximal VP pressure at a threshold value of 144.0 mmHg, dysphagia was identified with 96.4% sensitivity and 74.7% specificity. With maximal TB pressure at a threshold value of 158.0 mmHg, dysphagia was identified with 96.4% sensitivity and 77.3% specificity. At a threshold value of 2.0 mmHg for UES minimal pressure, dysphagia was diagnosed at 74.7% sensitivity and 60.7% specificity. Lastly, UES relaxation duration of <0.58 seconds had 85.7% sensitivity and 65.3% specificity, and UES resting pressure of <75.0 mmHg had 89.3% sensitivity and 90.7% specificity for identifying dysphagia. We present evidence that HRM could be a useful evaluation tool for detecting pharyngeal dysphagia.

Innovative human resource practices in U.S. hospitals: an empirical study.

PubMed

Platonova, Elena A; Hernandez, S Robert

2013-01-01

Contemporary organizations increasingly recognize human resource (HR) capabilities as a source of sustained competitive advantage; about 80% of an organization's value is attributable to intangible assets, including human assets and capital. Some scholars consider effective human resource management (HRM) the single most important factor affecting organizational performance. This study examined (1) the extent to which HRM strategies were included in organizational strategic planning and (2) the association between the involvement of senior HR professionals in strategic planning and the use of innovative HR practices in U.S. hospitals employing strategic HRM theory. A survey was administered to 168 chief executive officers and HR executives from 85 hospitals during spring 2005. Binary logistic regression was conducted to determine whether HRM involvement was associated with the use of innovative HRM strategies in the hospitals. We found significant associations between HRM strategy inclusion in the strategic planning process and senior HR professionals' involvement in organizational strategic planning and in three innovative HR activities: finding talent in advance for key job openings (odds ratio [OR] = 4.61, 95% confidence interval [CI]: 1.10-7.38), stressing organizational culture and values in the selection process (OR = 3.97, 95% CI: 1.01-3.97), and basing individual or team compensation on goal-oriented results (OR = 6.17, 95% CI: 1.17-3.37). Our data indicate that innovative HR practices were underused in some U.S. hospitals despite their potential to improve overall hospital performance. Hospitals that emphasized effective HRM were more likely to use some of the innovative HR approaches. In this article, we discuss this research and the practical implications of the findings.

A Framework for Assessing Initial Organizational Development Training in the U.S. Navy and the U.S. Army

DTIC Science & Technology

1982-12-01

recommends the addition of a practical, Nhands onv student learning ’experience to the HRM Specialist curriculum.I# X atS oRAA1 ’ DTIC TAB Unannounaed...historical evolution of the Navy’s Human Resource Management ( HRM ) and the Army’s Organizational Effectiveness (OE) programs, including a review of the...learning experi- ence to the HRM Specialist curriculum. . .. TABLE OF CONTENTS I. INTRODUCTION ---------------------------------- 10 A. GENERAL

Application of Classification Models to Pharyngeal High-Resolution Manometry

ERIC Educational Resources Information Center

Mielens, Jason D.; Hoffman, Matthew R.; Ciucci, Michelle R.; McCulloch, Timothy M.; Jiang, Jack J.

2012-01-01

Purpose: The authors present 3 methods of performing pattern recognition on spatiotemporal plots produced by pharyngeal high-resolution manometry (HRM). Method: Classification models, including the artificial neural networks (ANNs) multilayer perceptron (MLP) and learning vector quantization (LVQ), as well as support vector machines (SVM), were…

Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates.

PubMed

Saeidabadi, Mohammad Sadegh; Nili, Hassan; Dadras, Habibollah; Sharifiyazdi, Hassan; Connolly, Joanne; Valcanis, Mary; Raidal, Shane; Ghorashi, Seyed Ali

2017-06-01

Consumption of poultry products contaminated with Salmonella is one of the major causes of foodborne diseases worldwide and therefore detection and differentiation of Salmonella spp. in poultry is important. In this study, oligonucleotide primers were designed from hemD gene and a PCR followed by high-resolution melt (HRM) curve analysis was developed for rapid differentiation of Salmonella isolates. Amplicons of 228 bp were generated from 16 different Salmonella reference strains and from 65 clinical field isolates mainly from poultry farms. HRM curve analysis of the amplicons differentiated Salmonella isolates and analysis of the nucleotide sequence of the amplicons from selected isolates revealed that each melting curve profile was related to a unique DNA sequence. The relationship between reference strains and tested specimens was also evaluated using a mathematical model without visual interpretation of HRM curves. In addition, the potential of the PCR-HRM curve analysis was evaluated for genotyping of additional Salmonella isolates from different avian species. The findings indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Salmonella isolates to determine the serovar/serotype.

Effects of Physical Impairment on Grenade Throwing and Weapon Loading Tasks

DTIC Science & Technology

2015-03-01

1 GOVT PRINTG OFC (PDF) A MALHOTRA 1 ARMY RSCH LABORATORY – HRED (PDF) RDRL HRM D T DAVIS BLDG 5400 RM C242 REDSTONE ARSENAL...5002 1 ARMY RSCH LABORATORY – HRED (PDF) RDRL HRM DG J RUBINSTEIN BLDG 333 PICATINNY ARSENAL NJ 07806-5000 1 ARMY RSCH LABORATORY...HRED (PDF) ARMC FIELD ELEMENT RDRL HRM CH C BURNS THIRD AVE BLDG 1467B RM 336 FORT KNOX KY 40121 1 ARMY RSCH LABORATORY – HRED

Impaired bolus clearance in combined high-resolution esophageal manometry and impedance measurement helps to differentiate between esophagogastric junction outflow obstruction and achalasia.

PubMed

Zizer, Eugen; Seufferlein, Thomas; Hänle, Mark Martin

2017-02-01

Introduction and aims  High-resolution esophageal manometry (HRM) has improved the diagnostic work-up of esophageal motility disorders. Simultaneous evaluation of bolus clearance delivers useful information about the function of tubular esophagus. We assessed bolus clearance in a combined HRM-impedance examination for esophagogastric junction outflow obstruction (EGJOO) in comparison to achalasia patients. The collected data were assessed in a retrospective analysis. Patients and methods  After gastroscopy excluded a mechanical esophageal or gastric obstruction, 142 consecutive patients underwent combined HRM-impedance examination. The assessment and interpretation of the manometry results were done according to the Chicago Classification of esophageal motility disorders v3.0. After classifying the motility disorder, the evaluation of bolus clearance was done according to published studies. Results  All patients with achalasia (n = 24) showed a significantly impaired bolus clearance (< 80 %). Patients with unaffected peristalsis (n = 56) or patients with EGJOO (n = 14) each showed impaired clearance in 7 %, respectively. The evidence of axial hernia was not associated with impaired clearance. Conclusion  Our results demonstrate a significant difference in impedance measurements between EGJOO and achalasia cases. This might be helpful as an additional tool to differentiate between achalasia and EGJOO patients. Furthermore, the role of the combined impedance-HRM investigation for early diagnosis of achalasia in "pre-achalasia" condition or in evaluation of potential progress of EGJOO to achalasia should be evaluated in a prospective study. © Georg Thieme Verlag KG Stuttgart · New York.

The association between systemic sclerosis disease manifestations and esophageal high-resolution manometry parameters

PubMed Central

Kimmel, Jessica N.; Carlson, Dustin A.; Hinchcliff, Monique; Carns, Mary A.; Aren, Kathleen A; Lee, Jungwha; Pandolfino, John E.

2016-01-01

Background/Aims We aimed to evaluate the associations between SSc-related systemic manifestations and esophageal function using high-resolution manometry (HRM). Methods Patients with SSc that had undergone HRM between 1/2004 and 9/2014 were identified and HRMs were analyzed according to the Chicago Classification. Clinical characteristics were identified via retrospective chart review and compared among motility diagnoses while adjusting for age, gender, race, and SSc-disease duration. Results 79 patients (85% female, ages 25–77) were included. Clinical characteristics were compared between patients with absent contractility (AC, n = 40), ineffective esophageal motility (IEM; n = 15), and normal motility (n = 19); the 5 remaining patients met criteria for other motility diagnoses. Groups differed in severity of skin involvement measured by the modified Rodnan skin score (0–51): AC (adjusted mean 12.6), IEM (4.4), normal (4.3), p = 0.043. Pulmonary function tests [percent predicted FVC and DLCO) were lower in AC (adjusted mean, FVC: 70.3, DLCO 51.1), than IEM (FVC: 92.0; DLCO: 76.9) and normal motility (FVC: 80.0; DLCO: 67.2), p-values 0.057 (FVC) and 0.007 (DLCO). Groups did not differ by SSc-disease duration, autoantibodies, or reported symptoms of dysphagia or reflux. Conclusions In patients with SSc, absent esophageal contractility on HRM was associated with increased skin disease severity and worse lung function. Obtaining HRM to identify SSc patients with more severe esophageal dysfunction could be considered to enable implementation of management strategies in patients potentially at risk for increased morbidity and mortality. PMID:26921101

Clinical, Endoscopic, and Radiologic Features of Three Subtypes of Achalasia, Classified Using High-Resolution Manometry

PubMed Central

Khan, Mohammed Q.; AlQaraawi, Abdullah; Al-Sohaibani, Fahad; Al-Kahtani, Khalid; Al-Ashgar, Hamad I.

2015-01-01

Background/Aims: High-resolution manometry (HRM) has improved the accuracy of manometry in detecting achalasia and determining its subtypes. However, the correlation of achalasia subtypes with clinical, endoscopic, and radiologic findings has not been assessed. We aimed to evaluate and compare the clinical, endoscopic, and fluoroscopy findings associated with three subtypes of achalasia using HRM. Patients and Methods: The retrospective clinical data, HRM, endoscopy, and radiologic findings were obtained from the medical records of untreated achalasia patients. Results: From 2011 to 2013, 374 patients underwent HRM. Fifty-two patients (14%) were diagnosed with achalasia, but only 32 (8.5%) of these patients had not received treatment and were therefore included in this study. The endoscopy results were normal in 28% of the patients, and a barium swallow was inconclusive in 31% of the achalasia patients. Ten patients (31%) were classified as having type I achalasia, 17 (53%) were classified as type II, and 5 (16%) were classified as type III. Among the three subtypes, type I patients were on average the youngest and had the longest history of dysphagia, mildest chest pain, most significant weight loss, and most dilated esophagus with residual food. Chest pain was most common in type III patients, and frequently had normal fluoroscopic and endoscopic results. Conclusion: The clinical, radiologic, and endoscopic findings were not significantly different between patients with type I and type II untreated achalasia. Type III patients had the most severe symptoms and were the most difficult to diagnose based on varied clinical, radiologic, and endoscopic findings. PMID:26021774

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes

PubMed Central

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-01-01

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both “Zheda 23” and “Zheda 83”. Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species. PMID:28891982

Metabolic Pathways and Networks Associated with Tobacco Use in Military Personnel

PubMed Central

Jones, Dean P.; Walker, Douglas I.; Uppal, Karan; Rohrbeck, Patricia; Mallon, Timothy M.; Go, Young-Mi

2016-01-01

Objective Use high-resolution metabolomics (HRM) to identify metabolic pathways and networks associated with tobacco use in military personnel. Methods Four hundred de-identified samples obtained from the Department of Defense Serum Repository were classified as tobacco users or non-users according to cotinine content. HRM and bioinformatic methods were used to determine pathways and networks associated with classification. Results Eighty individuals were classified as tobacco users compared to 320 non-users based on cotinine levels ≥10 ng/mL. Alterations in lipid and xenobiotic metabolism, and diverse effects on amino acid, sialic acid and purine and pyrimidine metabolism were observed. Importantly, network analysis showed broad effects on metabolic associations not simply linked to well-defined pathways. Conclusions Tobacco use has complex metabolic effects which must be considered in evaluation of deployment-associated environmental exposures in military personnel. PMID:27501098

Implementing Strategic Orientation

ERIC Educational Resources Information Center

Fischer, Arthur K.; Brownback, Sarah

2012-01-01

An HRM case dealing with problems and issues of setting up orientation programs which align with corporate strategy. Discussion concerns how such a case can be used to exhibit the alignment between HRM and business strategy.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

PubMed Central

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H.; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A.

2015-01-01

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates. PMID:26394042

Polarization observables in hard rescattering mechanism of deuteron photodisintegration

NASA Astrophysics Data System (ADS)

Sargsian, Misak M.

2004-05-01

Polarization properties of high energy photodisintegration of the deuteron are studied within the framework of the hard rescattering mechanism (HRM). In HRM, a quark of one nucleon knocked-out by the incoming photon rescatters with a quark of the other nucleon leading to the production of two nucleons with high relative momentum. Summation of all relevant quark rescattering amplitudes allows us to express the scattering amplitude of the reaction through the convolution of a hard photon-quark interaction vertex, the large angle p-n scattering amplitude and the low momentum deuteron wave function. Within HRM, it is demonstrated that the polarization observables in hard photodisintegration of the deuteron can be expressed through the five helicity amplitudes of NN scattering at high momentum transfer. At 90° CM scattering HRM predicts the dominance of the isovector channel of hard pn rescattering, and it explains the observed smallness of induced, Py and transfered, Cx polarizations without invoking the argument of helicity conservation. Namely, HRM predicts that Py and Cx are proportional to the φ5 helicity amplitude which vanishes at θcm=90° due to symmetry reasons. HRM predicts also a nonzero value for Cz in the helicity-conserving regime and a positive Σ asymmetry which is related to the dominance of the isovector channel in the hard reinteraction. We extend our calculations to the region where large polarization effects are observed in pp scattering as well as give predictions for angular dependences.

A novel technique for bedside anorectal manometry in humans.

PubMed

Bharucha, A E; Stroetz, R; Feuerhak, K; Szarka, L A; Zinsmeister, A R

2015-10-01

Currently, anorectal manometry (ARM), which is used to diagnose defecatory disorders and identify anal weakness in fecal incontinence (FI) is generally conducted in specialized laboratories. Our aims were to compare anorectal functions measured with high-resolution manometry (HRM) and a novel portable manometry device. Anal pressures at rest, during squeeze, and simulated evacuation, and rectal sensation were evaluated with portable and HRM in 20 healthy women, 19 women with constipation, and 11 with FI. The relationship between anal pressures measured with portable and HRM was assessed by the concordance correlation coefficient (CCC), Bland Altman test, and paired t-tests. Anal pressures at rest (CCC 0.45; 95% CI: 0.29, 0.58) and during squeeze (CCC 0.60; 95% CI: 0.46, 0.72) measured with portable and HRM were correlated and inversely associated with the risk of FI. During simulated evacuation, the CCC for rectal pressure (0.62; 95% CI: 0.43, 0.76) was greater than that for anal pressure (CCC 0.22; 95% CI: 0.04, 0.39) and the rectoanal gradient (CCC 0.22; 95% CI: 0.02, 0.41). Rectal sensory thresholds for first sensation, the desire to defecate, and urgency measured by portable and HRM were also significantly correlated between techniques. For several parameters, differences between portable and HRM were statistically significant and the Bland Altman test was positive. Anorectal pressures and rectal sensation can be conveniently measured by portable manometry and are significantly correlated with high-resolution manometry. © 2015 John Wiley & Sons Ltd.

Development of cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers from the chloroplast genome of Glycyrrhiza species.

PubMed

Jo, Ick-Hyun; Sung, Jwakyung; Hong, Chi-Eun; Raveendar, Sebastin; Bang, Kyong-Hwan; Chung, Jong-Wook

2018-05-01

Licorice ( Glycyrrhiza glabra ) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species ( G. glabra and G. lepidota ). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2-4 genotypes) and melting curves (2-3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2-6 and 0.31-0.92, respectively. The genetic distance and polymorphism information content values were 0.16-0.76 and 0.15-0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.

Esophageal motor disorders in subjects with incidentally discovered Chagas disease: a study using high-resolution manometry and the Chicago classification.

PubMed

Remes-Troche, J M; Torres-Aguilera, M; Antonio-Cruz, K A; Vazquez-Jimenez, G; De-La-Cruz-Patiño, E

2014-08-01

In patients with chronic indeterminate Chagas disease, conventional manometry has shown that 25-48% had esophageal motor disorders. Recently, esophageal high-resolution manometry (HRM) has revolutionized the assessment of esophageal motor function. In this study, we performed esophageal HRM in a group of subjects with incidentally positive serological findings for Trypanosoma cruzi. In this prospective observational study, we evaluated subjects who had positive serological tests for Chagas disease detected during a screening evaluation for blood donation. All subjects underwent symptomatic evaluation and esophageal HRM with a 36 solid-state catheter. Esophageal abnormalities were classified using the Chicago classification. Forty-two healthy subjects (38 males) aged 18-61 years (mean age, 40.7 years) were included. When specific symptoms questionnaire was applied, 14 (33%) subjects had esophageal symptoms. Esophageal high-resolution manometry revealed that 28 (66%) of the subjects had an esophageal motility disorder according to the Chicago classification. Most common findings were hypocontractile disorders in 18 subjects (43%) and esophagogastric junction (EGJ) outflow obstruction in 6 (15%). Esophageal high-resolution manometry reveals that up to two thirds of the subjects with an incidental diagnosis of Chagas disease have esophageal abnormalities. This technology increases the detection and allows a more complete assessment of esophageal motor function in subjects infected with T. cruzi even in the early stages of the disease. © 2012 Copyright the Authors. Journal compilation © 2012, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.

Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards.

PubMed

Masny, Aleksander; Jagiełło, Agata; Płucienniczak, Grażyna; Golab, Elzbieta

2012-09-01

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus. Copyright © 2012 Elsevier B.V. All rights reserved.

Exploratory Research on Latent Esophageal Motility Disorders in Dysphagia Patients.

PubMed

Kawaguchi, Shinpei; Takeuchi, Toshihisa; Inoue, Yousuke; Takahashi, Yoshiaki; Ozaki, Haruhiko; Ota, Kazuhiro; Harada, Satoshi; Edogawa, Shoko; Kojima, Yuichi; Yamashita, Hiroshi; Fukuchi, Takumi; Ashida, Kiyoshi; Higuchi, Kazuhide

2017-01-01

High-resolution manometry (HRM) has been applied to assess esophageal motility disorders. However, the frequency and types of motility disorders in patients with dysphagia, which are frequently seen in clinical practice, are not clear. We evaluated latent esophageal motility disorders associated with dysphagia. The study included patients without erosive esophageal mucosal damage and with dysphagia symptoms refractory to at least 8 weeks of standard-dose proton pump inhibitors. After enrolment, HRM was used to evaluate for esophageal motility disorder based on the Chicago classification. Esophageal motility disorder was found in 58 of 100 patients and was classified based on the causes: achalasia (13%), esophagogastric junction outflow obstruction (16%), distal esophageal spasms (3%), weak peristalsis (14%), frequently failed peristalsis (5%), and hypertensive peristalsis (7%). Primary esophageal motility disorder was found in approximately 50% of cases in dysphagia patients. Therefore, esophageal motility disorder is not an uncommon condition and should be sought for in order to elucidate precisely the cause of dysphagia. © 2017 S. Karger AG, Basel.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

PubMed

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S

2016-01-01

Botrytis cinerea , is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdh B subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdh B mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdh B mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

PubMed Central

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S.

2016-01-01

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides. PMID:27895633

Differentiation of BHV-1 isolates from vaccine virus by high-resolution melting analysis.

PubMed

Ostertag-Hill, Claire; Fang, Liang; Izume, Satoko; Lee, Megan; Reed, Aimee; Jin, Ling

2015-02-16

An efficacious bovine herpesvirus type-1 (BHV-1) vaccine has been used for many years. However, in the past few years, abortion and respiratory diseases have occurred after administration of the modified live vaccine. To investigate whether BHV-1 isolates from disease outbreaks are identical to those of the vaccines used, selected regions of the BHV-1 genome were investigated by high-resolution melting (HRM) analysis and PCR-DNA sequencing. When a target region within the thymidine kinase (TK) gene was examined by HRM analysis, 6 out of the 11 isolates from abortion cases and 22 out of the 25 isolates from bovine respiratory disease (BRD) cases had different melting curves compared to the vaccine virus. Surprisingly, when a conserved region within the US6 gene that encodes glycoprotein D (gD) was examined by HRM analysis, 5 out of the 11 abortion isolates and 18 out of the 23 BRD isolates had different melting curves from the vaccine virus. To determine whether SNPs within the coding regions of glycoprotein E (gE) and TK genes can be used to differentiate the isolates from the vaccine virus, PCR-DNA sequencing was used to examine these SNPs in all the isolates. This revealed that only 1 out of 11 of the abortion isolates and 4 out of 24 of the BRD isolates are different in the target region of gE from the vaccine virus, while 5 out of 11 abortion isolates and 4 out of 22 BRD isolates are different in the target region of TK from the vaccine virus. No DNA sequence differences were observed in glycoprotein G (gG) region between disease and vaccine isolates. Our study demonstrated that many disease isolates had genetic differences from the vaccine virus in regions examined by HRM and PCR-DNA sequencing analysis. In addition, many isolates contained more than one type of mutation and were composed of mixed variants. Our study suggests that a mixture of variants were present in isolates collected post-vaccination. HRM is a rapid diagnostic method that can be used for rapid differentiation of clinical isolates from vaccine strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Diagnosis of genetic predisposition for lactose intolerance by high resolution melting analysis.

PubMed

Delacour, Hervé; Leduc, Amandine; Louçano-Perdriat, Andréa; Plantamura, Julie; Ceppa, Franck

2017-02-01

Lactose, the principle sugar in milk, is a disaccharide hydrolyzed by intestinal lactase into glucose and galactose, which are absorbed directly by diffusion in the intestine. The decline of lactase expression (or hypolactasia) in intestinal microvilli after weaning is a normal phenomenon in mammals known as lactase deficiency. It is observed in nearly 75% of the world population and is an inherited autosomal recessive trait with incomplete penetrance. It is caused by SNPs in a regulatory element for lactase gene. In Indo-European, lactase deficiency is associated with rs4982235 SNP (or -13910C>T). The aim of this study is to describe a method based on high resolution melting for rapidly detecting genetic predisposition to lactose intolerance. Analytical performance of the assay was assessed by evaluating within and betwwen-run precision and by comparing the results (n = 50 patients) obtained with the HRM assay to those obtained with the gold standard (Sanger sequencing of the region of interest). In silico prediction of HRM curves was performed to evaluate the potential impact of the other SNPs described within the PCR product on the HRM analytical performances. The assay has good performance (CV <0.2% during the between-run study). A perfect agreement with the gold standard method was observed. The presence of other polymorphisms within the amplified sequence is detected, the misclassification risk is low. This assay can be used for rapidly diagnosing genetic predisposition to lactose intolerance.

Association of High-Resolution Manometry Metrics with the Symptoms of Achalasia and the Symptomatic Outcomes of Peroral Esophageal Myotomy.

PubMed

Tang, Yurong; Xie, Chen; Wang, Meifeng; Jiang, Liuqin; Shi, Ruihua; Lin, Lin

2015-01-01

High-resolution manometry (HRM) has improved the accuracy of manometry in detecting achalasia and has helped distinguish between clinically relevant subtypes. This study investigated whether HRM metrics correlate with the achalasia symptoms and symptomatic outcomes of peroral esophageal myotomy (POEM). Of the 30 patients who were enrolled, 25 were treated with POEM, 12 of who underwent HRM after 3 months. All the patients completed the Eckardt score questionnaires, and those who underwent POEM were followed up for about 6 months. Pearson correlation was used to assess the relationship between the HRM metrics and symptoms and outcomes. The integrated relaxation pressure (IRP) score positively correlated with the total Eckardt score, regurgitation score and weight loss score in all the patients, and with the weight loss score in type I achalasia. In 25 patients (10 patients, type I; 15 patients, type II) who underwent POEM, the total Eckardt scores and individual symptom scores significantly decreased after surgery. Changes in the Eckardt scores were similar between type I and type II. Further, the Eckardt scores and weight loss score changes were positively correlated with baseline IRP. Twelve patients (4 patients, type I; 8 patients, type II) underwent HRM again after POEM. IRP changed significantly after POEM, as did the DEP in type II. The IRP changes after POEM were positively correlated with the Eckardt score changes. IRP is correlated with the symptoms and outcomes of achalasia patients. Thus, HRM is effective for assessing the severity of achalasia and can predict the efficacy of POEM.

Retaining Staff Employees: The Relationship between Human Resources Management Strategies and Organizational Commitment.

ERIC Educational Resources Information Center

Buck, Jeffrey M.; Watson, John L.

2002-01-01

Explored whether an institution's human resources management (HRM) strategies can influence individuals' organizational commitment levels, which ultimately can affect staff turnover rates. Found significant relationship between certain HRM strategies and commitment constructs. (EV)

Failed Deglutitive Upper Esophageal Sphincter Relaxation Is a Risk Factor for Aspiration in Stroke Patients with Oropharyngeal Dysphagia

PubMed Central

Lee, Taeheon; Park, Jung Ho; Sohn, Chongil; Yoon, Kyung Jae; Lee, Yong-Taek; Park, Jung Hwan; Jung, Il Seok

2017-01-01

Background/Aims We attempted to examine the relationship between abnormal findings on high-resolution manometry (HRM) and videofluoroscopic swallowing study (VFSS) of the pharynx and upper esophageal sphincter (UES), and to identify the risk factors for aspiration. Methods We performed VFSS and HRM on the same day in 36 ischemic stroke patients (mean age, 67.5 years) with dysphagia. Pressure (basal, median intra bolus, and nadir), relaxation time interval of the UES, and mesopharyngeal and hypopharyngeal contractility (as a contractile integral) were examined using HRM. The parameters of VFSS were vallecular residue, pyriform sinus residue, vallecular overflow, penetration, and aspiration. The association between the parameters of VFSS and HRM was analyzed by the Student’s t test. Results Three (8.3%) and 4 (11.1%) stroke patients with dysphagia had pyriform sinus residue and vallecular sinus residue, respectively, and 5 (13.8%) patients showed aspiration. Mesopharyngeal and hypopharyngeal contractile integrals in patients with residue in the pyriform sinus were significantly lower than those in patients without residue in the pyriform sinus (P < 0.05). Relaxation time intervals in patients with aspiration were significantly shorter than those in patients without aspiration (P < 0.05), and multivariate regression analysis revealed a shorter relaxation time interval as the main risk factor for aspiration (OR, 0.03; 95% CI, 0.01–0.65; P < 0.05). Conclusions Manometric measurements of the pharynx and UES were well correlated with abnormal findings in the VFSS, and a shorter relaxation time interval of the UES during deglutition is an important parameter for the development of aspiration. PMID:27510474

Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

PubMed Central

Wiengkum, Thanatcha; Srithep, Sarinee; Chainoi, Isarapong; Singboottra, Panthong; Wongwiwatthananukit, Sanchai

2011-01-01

Background Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. Methods Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion. Results HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. Conclusions Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate. PMID:21779184

Disability Management: Organizational Diversity and Dutch Employment Policy

PubMed Central

Haafkens, Joke A.

2010-01-01

Introduction While Human Resource Managers (HRM) and line managers could play a significant role in the prevention of job-related problems and in promotion of early job-continuation, it is not clear wether the chronically ill workers are recognized as a group. Unlike some other groups, distinguished by gender, age or ethnicity, those with chronic illness are less distinct and may not be included in diversity management programs. The aim of this research is to address theory and evidence in literature about the topic, as well as to inquire whether chronic illness of the employees is ‘visible’ in practice. Methods For desk research, we used a systematic search strategy involving medical, statistical, management, and social science databases (Web of Science, MedLine, Pub Med, Psych Info, etc.). Research results are based on case studies conducted with the managers and HRM of government and commercial organizations between March 2007 and October 2008 and between October 2008 and April 2009. These case studies were based on open interviews and focus group sessions (for human resource departments) which were consequently analyzed using thematical analysis. For group sessions, we used concept mapping to collect information from two groups of HRM professionals and managers. Secondary analysis included thematic and content analysis of ‘best practice’ organizations carried out by the Dutch organization Gatekeeper. Conclusions We have discovered that the chronically ill employees are largely invisible to HRM practitioners, line managers who do not always have the right instruments for implementation of the European or national frameworks. Most practitioners are unaware of the impact of chronic illness in their organizations and in employees work life. PMID:20383740

High-resolution Manometry and Globus: Comparison of Globus, Gastroesophageal Reflux Disease and Normal Controls Using High-resolution Manometry

PubMed Central

Choi, Won Seok; Kim, Tae Wan; Kim, Ja Hyun; Lee, Sang Hyuk; Hur, Woon Je; Choe, Young Gil; Lee, Sang Hyuk; Park, Jung Ho

2013-01-01

Background/Aims Globus is a foreign body sense in the throat without dysphagia, odynophagia, esophageal motility disorders, or gastroesophageal reflux. The etiology is unclear. Previous studies suggested that increased upper esophageal sphincter pressure, gastroesophageal reflux and hypertonicity of esophageal body were possible etiologies. This study was to quantify the upper esophageal sphincter (UES) pressure, contractile front velocity (CFV), proximal contractile integral (PCI), distal contractile integral (DCI) and transition zone (TZ) in patient with globus gastroesophageal reflux disease (GERD) without globus, and normal controls to suggest the correlation of specific high-resolution manometry (HRM) findings and globus. Methods Fifty-seven globus patients, 24 GERD patients and 7 normal controls were studied with HRM since 2009. We reviewed the reports, and selected 5 swallowing plots suitable for analysis in each report, analyzed each individual plot with ManoView. The 5 parameters from each plot in 57 globus patients were compared with that of 24 GERD patients and 7 normal controls. Results There was no significant difference in the UES pressure, CFV, PCI and DCI. TZ (using 30 mmHg isobaric contour) in globus showed significant difference compared with normal controls and GERD patients. The median values of TZ were 4.26 cm (interquartile range [IQR], 2.30-5.85) in globus patients, 5.91 cm (IQR, 3.97-7.62) in GERD patients and 2.26 cm (IQR, 1.22-2.92) in normal controls (P = 0.001). Conclusions HRM analysis suggested that UES pressure, CFV, PCI and DCI were not associated with globus. Instead increased length of TZ may be correlated with globus. Further study comparing HRM results in globus patients within larger population needs to confirm their correlation. PMID:24199007

Larvicidal Activities of Indigenous Bacillus thuringiensis Isolates and Nematode Symbiotic Bacterial Toxins against the Mosquito Vector, Culex pipiens (Diptera: Culicidae)

PubMed Central

Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A

2017-01-01

Background: The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Methods: Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens. B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC50 and LC95) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Results: Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6–5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Conclusion: Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases. PMID:29062851

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice.

PubMed

Chandler, Joshua D; Hu, Xin; Ko, Eun-Ju; Park, Soojin; Lee, Young-Tae; Orr, Michael; Fernandes, Jolyn; Uppal, Karan; Kang, Sang-Moo; Jones, Dean P; Go, Young-Mi

2016-11-01

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation. Copyright © 2016 the American Physiological Society.

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice

PubMed Central

Chandler, Joshua D.; Hu, Xin; Ko, Eun-Ju; Park, Soojin; Lee, Young-Tae; Orr, Michael; Fernandes, Jolyn; Uppal, Karan; Kang, Sang-Moo; Jones, Dean P.

2016-01-01

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation. PMID:27558316

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

A new "loyalty rewards" program in health care customer relationships.

PubMed

Macstravic, Scott

2006-01-01

"Loyalty rewards" in sponsored DM and HRM programs can apply to both providers and consumers. Physicians and hospitals can be paid to "loyally" adhere to payers' guidelines for managing diseases and risks. Many payer and their outsourced vendor programs include significant efforts to create collaborations between payer and provider, rather than relying on unilateral efforts. And growing numbers are rewarding providers for their efforts and results achieved.

Cultural Development through Human Resource Systems Integration.

ERIC Educational Resources Information Center

Albert, Michael

1985-01-01

Discusses the framework for developing a cultural human resources management (HRM) perspective. Central to this framework is modifying HRM programs to reinforce the organization's preferred practices. Modification occurs through selection, orientation, training and development, performance appraisal, career development, and compensation and…

(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione Prevents UVB-Induced Melanogenesis and Wrinkle Formation through Suppressing Oxidative Stress in HRM-2 Hairless Mice

PubMed Central

Lee, Bonggi; Moon, Kyoung Mi; Kim, Seong Jin; Kim, So Hee; Kim, Dae Hyun; An, Hye Jin; Jeong, Ji Won; Kim, Ye Ra; Son, Sujin; Kim, Min Jo; Chung, Ki Wung; Lee, Eun Kyeong; Chun, Pusoon; Ha, Young Mi; Kim, Min-Sun; Mo, Sang Hyun; Moon, Hyung Ryong; Chung, Hae Young

2016-01-01

Background. Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging. Our previous studies demonstrated that (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) inhibited tyrosinase activity and melanogenesis in vitro. Objective. To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkle formation, we topically applied MHY498 on dorsal skin of HRM-2 hairless mice. Methods. Using histological analysis, we evaluated effects of MHY498 on melanogenesis and wrinkle formation after UVB exposure. In addition, related molecular signaling pathways were examined using western blotting, fluorometric assay, and enzyme-linked immunosorbent assay. Results. MHY498 suppressed UVB-induced melanogenesis by inhibiting phosphorylation of CREB and translocation of MITF protein into the nucleus, which are key factors for tyrosinase expression. Consistently, tyrosinase protein levels were notably reduced in the dorsal skin of the hairless mice by MHY498 treatment. Furthermore, MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destruction by increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs, which play an essential role in collagen fiber degradation. As a mechanism, MHY498 notably ameliorated UVB-induced oxidative stress and NF-κB activation in the dermal skin of the hairless mice. Conclusion. Our study suggests that MHY498 can be used as a therapeutic or cosmetic agent for preventing uncontrolled melanogenesis and wrinkle formation. PMID:27242917

External electro-optic sampling utilizing a poled polymer asymmetric Fabry Perot cavity as an electro-optical probe tip

NASA Astrophysics Data System (ADS)

Chen, Kaixin; Zhang, Hongbo; Zhang, Daming; Yang, Han; Yi, Maobin

2002-09-01

External electro-optic sampling utilizing a poled polymer asymmetry Fabry-Perot cavity as electro-optic probe tip has been demonstrated. Electro-optical polymer spin coated on the high-reflectivity mirror (HRM) was corona poled. Thus, an asymmetric F-P cavity was formed based on the different reflectivity of the polymer and HRM and it converted the phase modulation that originates from electro-optic effect of the poled polymer to amplitude modulation, so only one laser beam is needed in this system. The principle of the sampling was analyzed by multiple reflection and index ellipsoid methods. A 1.2 GHz microwave signal propagating on coplanar waveguide transmission line was sampled, and the voltage sensitivity about 0.5 mV/ Hz was obtained.

Assessment of genetic mutations in the XRCC2 coding region by high resolution melting curve analysis and the risk of differentiated thyroid carcinoma in Iran

PubMed Central

Fayaz, Shima; Fard-Esfahani, Pezhman; Fard-Esfahani, Armaghan; Mostafavi, Ehsan; Meshkani, Reza; Mirmiranpour, Hossein; Khaghani, Shahnaz

2012-01-01

Homologous recombination (HR) is the major pathway for repairing double strand breaks (DSBs) in eukaryotes and XRCC2 is an essential component of the HR repair machinery. To evaluate the potential role of mutations in gene repair by HR in individuals susceptible to differentiated thyroid carcinoma (DTC) we used high resolution melting (HRM) analysis, a recently introduced method for detecting mutations, to examine the entire XRCC2 coding region in an Iranian population. HRM analysis was used to screen for mutations in three XRCC2 coding regions in 50 patients and 50 controls. There was no variation in the HRM curves obtained from the analysis of exons 1 and 2 in the case and control groups. In exon 3, an Arg188His polymorphism (rs3218536) was detected as a new melting curve group (OR: 1.46; 95%CI: 0.432–4.969; p = 0.38) compared with the normal melting curve. We also found a new Ser150Arg polymorphism in exon 3 of the control group. These findings suggest that genetic variations in the XRCC2 coding region have no potential effects on susceptibility to DTC. However, further studies with larger populations are required to confirm this conclusion. PMID:22481871

Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

NASA Astrophysics Data System (ADS)

Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

2017-02-01

In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

PubMed

Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

2014-02-01

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Quantifying esophagogastric junction contractility with a novel HRM topographic metric, the EGJ-Contractile Integral: normative values and preliminary evaluation in PPI non-responders.

PubMed

Nicodème, F; Pipa-Muniz, M; Khanna, K; Kahrilas, P J; Pandolfino, J E

2014-03-01

Despite its obvious pathophysiological relevance, the clinical utility of measures of esophagogastric junction (EGJ) contractility is unsubstantiated. High-resolution manometry (HRM) may improve upon this with its inherent ability to integrate the magnitude of contractility over time and length of the EGJ. This study aimed to develop a novel HRM metric summarizing EGJ contractility and test its ability distinguish among subgroups of proton pump inhibitor non-responders (PPI-NRs). 75 normal controls and 88 PPI-NRs were studied. All underwent HRM. PPI-NRs underwent pH-impedance monitoring on PPI therapy scored in terms of acid exposure, number of reflux events, and reflux-symptom correlation and grouped as meeting all criteria, some criteria, or no criteria of abnormality. Control HRM studies were used to establish normal values for candidate EGJ contractility metrics, which were then compared in their ability to differentiate among PPI-NR subgroups. The EGJ contractile integral (EGJ-CI), a metric integrating contractility across the EGJ for three respiratory cycles, best distinguished the All Criteria PPI-NR subgroup from controls and other PPI-NR subgroups. Normal values (median, [IQR]) for this measure were 39 mmHg-cm [25-55 mmHg-cm]. The correlation between the EGJ-CI and a previously proposed metric, the lower esophageal sphincter-pressure integral, that used a fixed 10 s time frame and an atmospheric as opposed to gastric pressure reference was weak. Among HRM metrics tested, the EGJ-CI was best in distinguishing PPI-NRs meeting all criteria of abnormality on pH-impedance testing. Future prospective studies are required to explore its utility in management of broader groups of gastroesophageal reflux disease patients. © 2013 John Wiley & Sons Ltd.

Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification.

PubMed

Duyvejonck, Hans; Cools, Piet; Decruyenaere, Johan; Roelens, Kristien; Noens, Lucien; Vermeulen, Stefan; Claeys, Geert; Decat, Ellen; Van Mechelen, Els; Vaneechoutte, Mario

2015-01-01

Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.

Estimating areas threatened by contamination from leaking chemical warfare agents dumped into the Baltic Sea

NASA Astrophysics Data System (ADS)

Jakacki, Jaromir; Przyborska, Anna; Andrzejewski, Jan

2017-04-01

Approximately 60,000 tons of chemical munitions were dumped into the Baltic Sea after World War II (the exact amount is unknown and some sources estimate it as more than 200,000 tons). Dumped munitions still pose a risk of leakage caused by erosion and corrosion, and it is important to know the danger areas. Because of wide dispersion of the dumped munitions, modelling is only one tool that could provide wide image of physical state of the sea at all locations and which could also be used for analysing contamination during a potential leakage. Obviously, it is possible to take samples at each dumpsite, but modelling also allows to develop possible scenarios of leakages under specific physical conditions. For the purpose of analysis of potential leakage a high-resolution model (HRM) of the contamination will be embedded in the hydrodynamic model (HM) of the Baltic Sea. The HRM will use data from general circulation model results of estimated resolution of nearly 2 km. The Parallel Ocean Program will be implemented as the HM for the whole Baltic Sea. Atmospheric data from regional implementation of the Weather Research and Forecasting System (WRF) have been used as the top boundary conditions of the HM, and sea level data from Gothenburg had been included into model barotropic equation as lateral boundary conditions. Passive tracer will represent the contamination in the HRM and horizontal resolution of the HRM will be close to 50 meters. Passive tracers will also be implemented in the HM - for comparison of the results. For proper representation of potential leakage of chemical warfare agents the HRM will have included diffusion and advection processes. The results from the HM are going to be interpolated into the HRM domain and then integration will be performed. Based on the implemented simulations, estimated contaminated area and its comparison from the HRM as well as from the HM will be presented. The research work was fund by the European Union (European Regional Development Fund) under the Interreg Baltic Sea Region Programme 2014-2020, project #R013 DAIMON (Decision Aid for Marine Munitions).

High-resolution Anorectal Manometry for Identifying Defecatory Disorders and Rectal Structural Abnormalities in Women.

PubMed

Prichard, David O; Lee, Taehee; Parthasarathy, Gopanandan; Fletcher, Joel G; Zinsmeister, Alan R; Bharucha, Adil E

2017-03-01

Contrary to conventional wisdom, the rectoanal gradient during evacuation is negative in many healthy people, undermining the utility of anorectal high-resolution manometry (HRM) for diagnosing defecatory disorders. We aimed to compare HRM and magnetic resonance imaging (MRI) for assessing rectal evacuation and structural abnormalities. We performed a retrospective analysis of 118 patients (all female; 51 with constipation, 48 with fecal incontinence, and 19 with rectal prolapse; age, 53 ± 1 years) assessed by HRM, the rectal balloon expulsion test (BET), and MRI at Mayo Clinic, Rochester, Minnesota, from February 2011 through March 2013. Thirty healthy asymptomatic women (age, 37 ± 2 years) served as controls. We used principal components analysis of HRM variables to identify rectoanal pressure patterns associated with rectal prolapse and phenotypes of patients with prolapse. Compared with patients with normal findings from the rectal BET, patients with an abnormal BET had lower median rectal pressure (36 vs 22 mm Hg, P = .002), a more negative median rectoanal gradient (-6 vs -29 mm Hg, P = .006) during evacuation, and a lower proportion of evacuation on the basis of MRI analysis (median of 40% vs 80%, P < .0001). A score derived from rectal pressure and anorectal descent during evacuation and a patulous anal canal was associated (P = .005) with large rectoceles (3 cm or larger). A principal component (PC) logistic model discriminated between patients with and without prolapse with 96% accuracy. Among patients with prolapse, there were 2 phenotypes, which were characterized by high (PC1) or low (PC2) anal pressures at rest and squeeze along with higher rectal and anal pressures (PC1) or a higher rectoanal gradient during evacuation (PC2). In a retrospective analysis of patients assessed by HRM, measurements of rectal evacuation by anorectal HRM, BET, and MRI were correlated. HRM alone and together with anorectal descent during evacuation may identify rectal prolapse and large rectoceles, respectively, and also identify unique phenotypes of rectal prolapse. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Applying Organization Development to Coast Guard Affirmative Action for Black Officers.

DTIC Science & Technology

1979-12-01

systems approach in the HRM area which includes significant attention to equal opportunity and affirmative action issues. Its present affirmative action...systems approach to the most funda- mental objective of managers - the acquisition and development of people - is needed, not only to meet soci.i and legal...strategy will not produce change, it will largely maintain the status quo. A total systems approach offers the hope of changing the present pattern

Organisation Learners' Competence to Overcome Organisation's Learning Inertia: A Conceptual Framework

ERIC Educational Resources Information Center

Arunprasad, P.

2015-01-01

This paper identifies the interrelationship between the organisational learning (OL) characteristics, strategic human resource management (HRM) practices and the corresponding learning outcome for a sustained competitive advantage (SCA). Through a profound literature review, first, the knowledge assets (human capital), OL and strategic HRM are…

Esophageal motor abnormalities in eosinophilic esophagitis identified by high-resolution manometry.

PubMed

Martín Martín, Leticia; Santander, Cecilio; Lopez Martín, Mari Carmen; Espinoza-Ríos, Jorge; Chavarría-Herbozo, Carlos; Gisbert, Javier P; Moreno-Otero, Ricardo

2011-09-01

Esophageal motility abnormalities, as measured by conventional manometry (CM), are non-specific in the majority of patients with eosinophilic esophagitis (EoE). Moreover, the study of CM is limited by poor interobserver agreement. The aims of the present study were: (i) to assess the esophageal patterns in EoE by a topographic analysis of high-resolution manometry (HRM) data; and (ii) to establish a relationship between motility abnormalities and symptoms of EoE, such as dysphagia and bolus impaction. All adult patients with EoE diagnosed according to histological criteria, and controls with gastroesophageal reflux disease symptoms and dysphagia, were included. HRM was done in EoE patients and controls. For the analysis of data, the Chicago classification was followed. HRM was performed in 21 patients with EoE, as well as in 21 controls. Of the 21 patients with EoE, 10 (48%) showed pan-esophageal pressurization, six (28%) showed peristaltic dysfunction, and in five cases (24%), HRM was normal. There was no pan-esophageal pressurization in controls. Nine of 10 patients with pan-esophageal pressurization required endoscopic bolus removal (P < 0.05); none had obstructive endoscopy findings. The most frequent esophageal motor abnormality measured by HRM was a pan-esophageal pressurization. Bolus impaction in patients with EoE was associated with pan-esophageal pressurization. © 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

PubMed

Fraley, Stephanie I; Hardick, Justin; Masek, Billie J; Jo Masek, Billie; Athamanolap, Pornpat; Rothman, Richard E; Gaydos, Charlotte A; Carroll, Karen C; Wakefield, Teresa; Wang, Tza-Huei; Yang, Samuel

2013-10-01

Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

PubMed

Ferro, Marta; Macher, Hada C; Noguerol, Pilar; Jimenez-Arriscado, Pilar; Molinero, Patrocinio; Guerrero, Juan M; Rubio, Amalia

2016-01-01

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.

Value of PAX1 Methylation Analysis by MS-HRM in the Triage of Atypical Squamous Cells of Undetermined Significance.

PubMed

Li, Shi-Rong; Wang, Zhen-Ming; Wang, Yu-Hui; Wang, Xi-Bo; Zhao, Jian-Qiang; Xue, Hai-Bin; Jiang, Fu-Guo

2015-01-01

Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chi- square test was used to test the differences of PAX1 methylation or HPV infection between groups. The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.

Hard Break-Up of Two-Nucleons and QCD Dynamics of NN Interaction

NASA Astrophysics Data System (ADS)

Sargsian, Misak; Granados, Carlos

2009-05-01

We investigate hard photodisintegration of two nucleons from ^3He nucleus within the framework of hard rescattering model (HRM). In HRM a quark of one nucleon knocked-out by incoming photon rescatters with a quark of the other nucleon leading to the production of two nucleons with high relative momentum. HRM allows to express the amplitude of two-nucleon break-up reaction through the convolution of photon-quark scattering, NN hard scattering amplitude and nuclear spectral function which can be calculated using nonrelativistic ^3He wave function. HRM predicts several specific features for hard break-up reaction. First, the cross section will approximately scale as s-11. Also one predicts comparable or larger cross section for pp break up as compared to that of pn break-up, which is opposite to what is observed in low energy kinematics. Another result is the prediction of different spectator momentum dependencies of pp and pn break-up cross sections. This is due to the fact that same-helicity pp-component is strongly suppressed in the ground state wave function of ^3He. Due to this suppression HRM predicts significantly different asymmetries for the cross section of polarization transfer NN break-up reactions for circularly polarized photons. For the pp break-up this asymmetry is predicted to be zero while for the pn it is close to 23.

High-resolution Melting Analysis for Gene Scanning of Adenomatous Polyposis Coli (APC) Gene With Oral Squamous Cell Carcinoma Samples.

PubMed

Chang, Ya-Sian; Lin, Chien-Yu; Yang, Shu-Fen; Ho, Cheng Mao; Chang, Jan-Gowth

2016-02-01

There have been many different mutations reported for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor suppressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and susceptibility to oral squamous cell carcinoma (OSCC). High-resolution melting (HRM) analysis was used to characterize APC mutations. Genomic DNA was extracted from 83 patient specimens of OSCC and 50 blood samples from healthy control subjects. The 14 exons and mutation cluster region of exon 15 were screened by HRM analysis. All mutations were confirmed by direct DNA sequencing. Three mutations and 4 single nucleotide polymorphisms (SNPs) were found in this study. The mutations were c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, and c.1488A>T (T496T) in exon 11. Two SNPs, c.4479G>A (T1493T) and c.5465A>T (D1822V), were located in exon 15, whereas c.1458T>C (Y486Y) and c.1635G>A (A545A) were located in exon 11 and 13, respectively. There was no observed association between OSCC risk and genotype for any of the 4 APC SNPs. The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

Accuracy of the Garmin 920 XT HRM to perform HRV analysis.

PubMed

Cassirame, Johan; Vanhaesebrouck, Romain; Chevrolat, Simon; Mourot, Laurent

2017-12-01

Heart rate variability (HRV) analysis is widely used to investigate autonomous cardiac drive. This method requires periodogram measurement, which can be obtained by an electrocardiogram (ECG) or from a heart rate monitor (HRM), e.g. the Garmin 920 XT device. The purpose of this investigation was to assess the accuracy of RR time series measurements from a Garmin 920 XT HRM as compared to a standard ECG, and to verify whether the measurements thus obtained are suitable for HRV analysis. RR time series were collected simultaneously with an ECG (Powerlab system, AD Instruments, Castell Hill, Australia) and a Garmin XT 920 in 11 healthy subjects during three conditions, namely in the supine position, the standing position and during moderate exercise. In a first step, we compared RR time series obtained with both tools using the Bland and Altman method to obtain the limits of agreement in all three conditions. In a second step, we compared the results of HRV analysis between the ECG RR time series and Garmin 920 XT series. Results show that the accuracy of this system is in accordance with the literature in terms of the limits of agreement. In the supine position, bias was 0.01, - 2.24, + 2.26 ms; in the standing position, - 0.01, - 3.12, + 3.11 ms respectively, and during exercise, - 0.01, - 4.43 and + 4.40 ms. Regarding HRV analysis, we did not find any difference for HRV analysis in the supine position, but the standing and exercise conditions both showed small modifications.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

2014-01-01

Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

Differentiation of minute virus of mice and mouse parvovirus by high resolution melting curve analysis.

PubMed

Rao, Dan; Wu, Miaoli; Wang, Jing; Yuan, Wen; Zhu, Yujun; Cong, Feng; Xu, Fengjiao; Lian, Yuexiao; Huang, Bihong; Wu, Qiwen; Chen, Meili; Zhang, Yu; Huang, Ren; Guo, Pengju

2017-12-01

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/μL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV. Copyright © 2017 Elsevier B.V. All rights reserved.

A Systems Model Comparing Australian and Chinese HRM Education

ERIC Educational Resources Information Center

Davidson, Paul; Tsakissiris, Jane; Guo, Yuanyuan

2017-01-01

This paper explores the implications for learning design in HRM education in the 21st century. An open systems perspective is used to argue the importance of establishing productive relationships between academia, professional associations, regulators and industry (resource inputs) to support the creation of optimal learning environments (the…

78 FR 8174 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-05

... Smaligo, Chairman, Building, 633 West team.com/lomrs.hrm. County (12-06- Tulsa County Board 3rd Street... flood insurance premium rates for new buildings and the contents of those buildings. For rating purposes....rampp- March 28, 2013 400360 Collinsville (12- Weaver, Mayor, Street, team.com/lomrs.hrm. 06-4005P...

Influence of HRM Practices on Organizational Commitment: A Study among Software Professionals in India

ERIC Educational Resources Information Center

Paul, A. K.; Anantharaman, R. N.

2004-01-01

Although organizational commitment has been discussed frequently in organizational psychology for almost four decades, few studies have involved software professionals. A study in India reveals that HRM practices such as employee-friendly work environment, career development, development oriented appraisal, and comprehensive training show a…

Contemplation and the "Performative Absolute": Submission and Identity in Managerial Modernity

ERIC Educational Resources Information Center

Roberts, Richard H.

2013-01-01

Practices derived from the "vita contemplativa" and other spiritual sources are drawn upon by management, but as the power of human resources management (HRM) is extended so the relationship between "contemplation" and the surrender of self-identity required by HRM demands critical examination. The conscious construction of the…

Challenges Implementing Work-Integrated Learning in Human Resource Management University Courses

ERIC Educational Resources Information Center

Rook, Laura

2017-01-01

The examination of work-integrated learning (WIL) programs in the undergraduate Human Resource Management (HRM) curriculum is an area under-represented in the Australian literature. This paper identifies the challenges faced in implementing WIL into the HRM undergraduate curriculum. Qualitative semi-structured interviews were conducted with 38…

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor.

PubMed

Dong, Chongmei; Vincent, Kate; Sharp, Peter

2009-12-04

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

PubMed Central

Cho, Young-Il; Ahn, Yul-Kyun; Tripathi, Swati; Kim, Jeong-Ho; Lee, Hye-Eun; Kim, Do-Sun

2015-01-01

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species. PMID:25790283

Esophageal testing: What we have so far

PubMed Central

de Bortoli, Nicola; Martinucci, Irene; Bertani, Lorenzo; Russo, Salvatore; Franchi, Riccardo; Furnari, Manuele; Tolone, Salvatore; Bodini, Giorgia; Bolognesi, Valeria; Bellini, Massimo; Savarino, Vincenzo; Marchi, Santino; Savarino, Edoardo Vincenzo

2016-01-01

Gastroesophageal reflux disease (GERD) is a common disorder of the gastrointestinal tract. In the last few decades, new technologies have evolved and have been applied to the functional study of the esophagus, allowing for the improvement of our knowledge of the pathophysiology of GERD. High-resolution manometry (HRM) permits greater understanding of the function of the esophagogastric junction and the risks associated with hiatal hernia. Moreover, HRM has been found to be more reproducible and sensitive than conventional water-perfused manometry to detect the presence of transient lower esophageal sphincter relaxation. Esophageal 24-h pH-metry with or without combined impedance is usually performed in patients with negative endoscopy and reflux symptoms who have a poor response to anti-reflux medical therapy to assess esophageal acid exposure and symptom-reflux correlations. In particular, esophageal 24-h impedance and pH monitoring can detect acid and non-acid reflux events. EndoFLIP is a recent technique poorly applied in clinical practice, although it provides a large amount of information about the esophagogastric junction. In the coming years, laryngopharyngeal symptoms could be evaluated with up and coming non-invasive or minimally invasive techniques, such as pepsin detection in saliva or pharyngeal pH-metry. Future studies are required of these techniques to evaluate their diagnostic accuracy and usefulness, although the available data are promising. PMID:26909230

Devolving HRM and HRD to Line managers: The Case of Hilton International's UK Hotels

ERIC Educational Resources Information Center

Maxwell, Gillian; Watson, Sandra

2004-01-01

This paper explores line manager responsibility for human resource management (HRM) and human resource development (HRD) in an international hotel organisation. The results identify a strong support system for line managers in relation to training and good relations with HR specialists. However, less evident is senior management support coupled…

HRM Practices in Public and Private Universities of Pakistan: A Comparative Study

ERIC Educational Resources Information Center

Iqbal, Muhammad Zafar; Arif, Muhammad Irfan; Abbas, Furrakh

2011-01-01

The purpose of this study was to compare the HRM practices of public and private universities in Punjab province of Pakistan. The data for the study was collected through a questionnaire comprising 30 items mainly related to job definition, training and development, compensation, team work, employee's participation and performance appraisal. The…

Strategic Role of HRM in Turkey: A Three-Country Comparative Analysis

ERIC Educational Resources Information Center

Ozcelik, Ayse Oya; Aydinli, Fulya

2006-01-01

Purpose: To explore the strategic role of human resource management (HRM) in Turkey by comparing Turkish companies to Spanish and German companies. Design/methodology/approach: The questionnaire form of the Cranet-G 1999-2000 Survey (Cranfield Network on Strategic International Human Resource Management) has been used to collect the data. The…

Human Resources Management & Development Handbook. Second Edition.

ERIC Educational Resources Information Center

Tracey, William R., Ed.

This revised handbook on the theory and practice of human resources management and development (HRM/D) focuses on people management and the personnel development processes. The book's 18 parts and 102 chapters by 107 contributors provide authoritative and comprehensive information on every aspect of modern HRM/D. Part 1 provides an overview of…

Strategic HRM for SMEs: Implications for Firms and Policy

ERIC Educational Resources Information Center

Brand, Maryse J.; Bax, Erik H.

2002-01-01

This paper is on the growing importance of strategic human resource management (SHRM) for small and medium-sized enterprises (SMEs). Many small firms encounter serious human resource problems, while at the same time these human resources play a vital role in developing and sustaining their competitive advantages. In (S)HRM literature specific…

Mapping Out Devolution: An Exploration of the Realities of Devolution

ERIC Educational Resources Information Center

Cascon-Pereira, Rosalia; Valverde, Mireia; Ryan, Gerard

2006-01-01

Purpose: Few definitions in the HRM literature have reached as much consensus as the term "devolution". However, devolution is a phenomenon that has been defined from the perspective of HRM specialists, with little or no contribution from middle managers. This paper seeks to explore what is behind the commonly shared definition of…

Competence and Employment Forum: Linking HRD and HRM

ERIC Educational Resources Information Center

Siikaniemi, Lena

2009-01-01

Purpose: The purpose of this paper is to find a model for the individual employee, the employer, the supervisors and the personnel at HR to manage organizational and individual changes. The objective of the model is to manage the changes in parallel with the competence (HRD) and employment (HRM) aspects. Design/methodology/approach: The research…

Real-time Continuous Esophageal High-resolution Manometry (HRM) During Laparoscopic Heller Myotomy and Dor Fundoplication for the Treatment of Achalasia. A Promising Novelty in Regards of Perfecting Surgical Technique: Could It Guide Surgical Technique Toward Excellent Results?

PubMed

Triantafyllou, Tania; Doulami, Georgia; Papailiou, Joanna; Mantides, Apostolos; Zografos, Georgios; Theodorou, Dimitrios

2016-12-01

High-resolution manometry (HRM) is the gold-standard diagnostic tool for achalasia of the esophagus. Laparoscopic Heller-Dor technique is the preferred surgical approach with success rate estimated 90%. The use of intraoperative HRM provides real-time estimation of intraluminal esophageal pressures and identifies the exact points of esophageal luminal pressure during laparoscopy. Ten patients with achalasia underwent surgery. All patients preoperatively completed 1 manometric study and Quality of Life questionnaires (EORTC QLQ-C30 version 3.0) with Eckardt scores. We collected intraoperative manometry data and repeated manometric studies, EORTC QLQ-C30, and Eckardt scores postoperatively. Median Eckardt score was decreased from 7.5 to 0.5, mean resting pressure decreased from 51.4 to 11.9 mm Hg, whereas mean residual pressure diminished from 45.9 to 9.5 mm Hg postoperatively. The simultaneous use of HRM during the Heller-Dor technique may lead to an individualized management of the disease.

Comparison of upright LBPP and supine LBNP in terms of cardiovascular and biomechanical parameters to simulate 1/6-G (lunar gravity) and 3/8-G (Martian gravity) activities

NASA Astrophysics Data System (ADS)

Schlabs, Thomas; Rosales-Velderrain, Armando; Ruckstuhl, Heidi; Richardson, Sara E.; Hargens, Alan

Background: Missions of astronauts to Moon and Mars may be planned in the future. From over 40 years of manned spaceflight it is known that the human body experiences cardiovascular and musculoskeletal losses and a decrease in aerobic fitness while exposed to reduced gravity. Because future missions will be much longer than before, further research is needed to improve Earth-based simulations of reduced gravity. Among others, two methods are capable of simu-lating fractional gravity on Earth: upright Lower Body Positive Pressure (LBPP) and supine Lower Body Negative Pressure (LBNP). No previous study has directly compared these two methods to determine which method is better suited to simulate both the biomechanical and cardiovascular responses of performing activity in lunar (1/6-G) and Martian (3/8-G) gravities. Taken previous studies into account and considering the fact that supine posture is closer to the established 10 head-up-tilt lunar simulation, we hypothesized that exercise performed in supine LBNP better simulates the cardiovascular conditions that occur in lunar and Martian gravities. Methods: 12 healthy normal subjects underwent a protocol consisting of resting and walking (0.25 Froude) with LBNP and LBPP. Each protocol was performed in simulated 1/6-G and 3/8-G. Heart-rate (HR), blood pressure, oxygen consumption (VO2), vertical component of the ground reaction force, comfort of the subject and perceived exertion of the subject (Borg Scale) were assessed. The obtained parameters were compared to predicted values for lunar and Martian gravity conditions in order to determine the method that shows the best level of agreement. Results: There was no difference in gait parameters between LBPP and LBNP simulation of lunar and Martian gravity (cadence: P=0.427, normalized stride length: P=0.373, duty fac-tor: P=0.302, and normalized vertical peak force (P=0.064). Mean blood pressure (P=0.398), comfort (P=0.832) and BORG rating (P=0.186) did not differ between the two methods. How-ever, we found that the heart rate (P=0.022) and VO2 (P=0.038) were significantly higher during LBPP (HRM oon =74±3 bpm, HRM ars =80±3 bpm, VO2M oon =4.6±0.22 l*kg-1 *min-1 , VO2M ars =6.17±0.38 l*kg-1 *min-1 ) than in LBNP (HRM oon =72±3 bpm, HRM ars =77±3 bpm, VO2M oon =4.66±0.43 l*kg-1 *min-1 , VO2M ars =5.66±0.42 l*kg-1 *min-1 ). A further analysis of deviation from the predicted parameters (HRM oon =77 bpm, HRM ars =84 bpm, VO2M oon =5.40 l*kg-1 *min-1 , VO2M ars =7.14 l*kg-1 *min-1 ) revealed a smaller deviation for LBPP (∆HR=4 bpm, ∆VO2=0.89 l*kg-1 *min-1 ) as compared to LBNP (∆HR=6 bpm, ∆VO2=1.11 l*kg-1 *min-1 ). Discussion: We conclude that biomechanical characteristics of gait are not different between supine LBNP and upright LBPP. In terms of cardiovascular parameters there are only differ-ences in heart rate and VO2. The higher heart rate during upright LBPP is probably due to a lower preload of the heart; as venous return is attenuated when the subject is positioned upright instead of supine. The higher VO2 during upright LBPP reflects the increased activity of anti-gravity muscles working to keep the upper body in balance without body suspension. Most skeletal muscles are not used when lying at rest in supine LBNP. Considering that values for heart rate and VO2 produced with the LBPP simulation had a smaller deviation from the predicted values than with the LBNP simulation, we conclude that upright LBPP is obviously better suited to simulate both lunar and Martian activities.

PAX1 methylation analysis by MS-HRM is useful in triage of high-grade squamous intraepithelial lesions.

PubMed

Wang, Zhen-Ming

2014-01-01

This study is aimed to investigate the role of paired boxed gene 1 (PAX1) methylation analysis by methylation- sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and compared its performance with the Hybrid Capture 2 (HC2) human papillomavirus (HPV) test. In our study, 130 cases with a diagnosis of ASC-H from the cervical cytological screening by Thinprep cytologic test (TCT) technique were selected for triage. Their cervical scrapings were collected and evaluated by using PAX1 methylation analysis (MS-HRM) and high-risk HPV DNA test (HC2), followed by colposcopy and cervical biopsy. Chi-square test were used to test the differences of PAX1 methylation or HPV infection between groups. In the detection of CIN2+, the sensitivity, specificity, the PPV, NPV and the accuracy of PAX1 MS-HRM assay and high-risk HPV (HR-HPV) tests were respectively 80.6% vs 67.7%, 94.9% vs 54.5%, 83.3%, vs 31.8%, 94.0% vs 84.4%, and 91.5% vs 57.7%. The PAX1 MS-HRM assay proved superior to HR-HPV testing in the detection of high grade lesions (CIN2+) in ASC-H. This approach could screen out the majority of high grade lesion cases of ASC-H, and thus could reduce the referral rate to colposcopy.

High-Resolution Melting-Curve Analysis of obg Gene to Differentiate the Temperature-Sensitive Mycoplasma synoviae Vaccine Strain MS-H from Non-Temperature-Sensitive Strains

PubMed Central

Shahid, Muhammad A.; Markham, Philip F.; Marenda, Marc S.; Agnew-Crumpton, Rebecca; Noormohammadi, Amir H.

2014-01-01

Temperature-sensitive (ts +) vaccine strain MS-H is the only live attenuated M. synoviae vaccine commercially available for use in poultry. With increasing use of this vaccine to control M. synoviae infections, differentiation of MS-H from field M. synoviae strains and from rarely occurring non-temperature-sensitive (ts –) MS-H revertants has become important, especially in countries where local strains are indistinguishable from MS-H by sequence analysis of variable lipoprotein haemagglutinin (vlhA) gene. Single nucleotide polymorphisms (SNPs) in the obg of MS-H have been found to associate with ts phenotype. In this study, four PCRs followed by high-resolution melting (HRM)-curve analysis of the regions encompassing these SNPs were developed and evaluated for their potential to differentiate MS-H from 36 M. synoviae strains/isolates. The nested-obg PCR-HRM differentiated ts + MS-H vaccine not only from field M. synoviae strains/isolates but also from ts – MS-H revertants. The mean genotype confidence percentages, 96.9±3.4 and 8.8±11.2 for ts + and ts – strains, respectively, demonstrated high differentiating power of the nested-obg PCR-HRM. Using a combination of nested-obg and obg-F3R3 PCR-HRM, 97% of the isolates/strains were typed according to their ts phenotype with all MS-H isolates typed as MS-H. A set of respiratory swabs from MS-H vaccinated specific pathogen free chickens and M. synoviae infected commercial chicken flocks were tested using obg PCR-HRM system and results were consistent with those of vlhA genotyping. The PCR-HRM system developed in this study, proved to be a rapid and reliable tool using pure M. synoviae cultures as well as direct clinical specimens. PMID:24643035

Investigation of Dysphagia After Antireflux Surgery by High-resolution Manometry: Impact of Multiple Water Swallows and a Solid Test Meal on Diagnosis, Management, and Clinical Outcome.

PubMed

Wang, Yu Tien; Tai, Ling Fung; Yazaki, Etsuro; Jafari, Jafar; Sweis, Rami; Tucker, Emily; Knowles, Kevin; Wright, Jeff; Ahmad, Saqib; Kasi, Madhavi; Hamlett, Katharine; Fox, Mark R; Sifrim, Daniel

2015-09-01

Management of patients with dysphagia, regurgitation, and related symptoms after antireflux surgery is challenging. This prospective, case-control study tested the hypothesis that compared with standard high-resolution manometry (HRM) with single water swallows (SWS), adding multiple water swallows (MWS) and a solid test meal increases diagnostic yield and clinical impact of physiological investigations. Fifty-seven symptomatic and 12 asymptomatic patients underwent HRM with SWS, MWS, and a solid test meal. Dysphagia and reflux were assessed by validated questionnaires. Diagnostic yield of standard and full HRM studies with 24-hour pH-impedance monitoring was compared. Pneumatic dilatation was performed for outlet obstruction on HRM studies. Clinical outcome was assessed by questionnaires and an analogue scale with "satisfactory" defined as at least 40% symptom improvement requiring no further treatment. Postoperative esophagogastric junction pressure was similar in all groups. Abnormal esophagogastric junction morphology (double high pressure band) was more common in symptomatic than in control patients (13 of 57 vs 0 of 12, P = .004). Diagnostic yield of HRM was 11 (19%), 11 (19%), and 33 of 57 (58%), with SWS, MWS, and solids, respectively (P < .001); it was greatest for solids in patients with dysphagia (19 of 27, 70%). Outlet obstruction was present in 4 (7%), 11 (19%), and 15 of 57 patients (26%) with SWS, MWS, and solids, respectively (P < .009). No asymptomatic control had clinically relevant dysfunction on solid swallows. Dilatation was performed in 12 of 15 patients with outlet obstruction during the test meal. Symptom response was satisfactory, good, or excellent in 7 of 12 (58%) with no serious complications. The addition of MWS and a solid test meal increases the diagnostic yield of HRM studies in patients with symptoms after fundoplication and identifies additional patients with outlet obstruction who benefit from endoscopic dilatation. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Development of COS-SNP and HRM markers for high-throughput and reliable haplotype-based detection of Lr14a in durum wheat (Triticum durum Desf.).

PubMed

Terracciano, Irma; Maccaferri, Marco; Bassi, Filippo; Mantovani, Paola; Sanguineti, Maria C; Salvi, Silvio; Simková, Hana; Doležel, Jaroslav; Massi, Andrea; Ammar, Karim; Kolmer, James; Tuberosa, Roberto

2013-04-01

Leaf rust (Puccinia triticina Eriks. & Henn.) is a major disease affecting durum wheat production. The Lr14a-resistant gene present in the durum wheat cv. Creso and its derivative cv. Colosseo is one of the best characterized leaf-rust resistance sources deployed in durum wheat breeding. Lr14a has been mapped close to the simple sequence repeat markers gwm146, gwm344 and wmc10 in the distal portion of the chromosome arm 7BL, a gene-dense region. The objectives of this study were: (1) to enrich the Lr14a region with single nucleotide polymorphisms (SNPs) and high-resolution melting (HRM)-based markers developed from conserved ortholog set (COS) genes and from sequenced Diversity Array Technology (DArT(®)) markers; (2) to further investigate the gene content and colinearity of this region with the Brachypodium and rice genomes. Ten new COS-SNP and five HRM markers were mapped within an 8.0 cM interval spanning Lr14a. Two HRM markers pinpointed the locus in an interval of <1.0 cM and eight COS-SNPs were mapped 2.1-4.1 cM distal to Lr14a. Each marker was tested for its capacity to predict the state of Lr14a alleles (in particular, Lr14-Creso associated to resistance) in a panel of durum wheat elite germplasm including 164 accessions. Two of the most informative markers were converted into KASPar(®) markers. Single assay markers ubw14 and wPt-4038-HRM designed for agarose gel electrophoresis/KASPar(®) assays and high-resolution melting analysis, respectively, as well as the double-marker combinations ubw14/ubw18, ubw14/ubw35 and wPt-4038-HRM-ubw35 will be useful for germplasm haplotyping and for molecular-assisted breeding.

New strategy for rapid diagnosis and characterization of fungal infections: the example of corneal scrapings.

PubMed

Goldschmidt, Pablo; Degorge, Sandrine; Che Sarria, Patricia; Benallaoua, Djida; Semoun, Oudy; Borderie, Vincent; Laroche, Laurent; Chaumeil, Christine

2012-01-01

The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction.

A Comparison of Two Measures of HIV Diversity in Multi-Assay Algorithms for HIV Incidence Estimation

PubMed Central

Cousins, Matthew M.; Konikoff, Jacob; Sabin, Devin; Khaki, Leila; Longosz, Andrew F.; Laeyendecker, Oliver; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Kobin, Beryl A.; Wheeler, Darrell; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Brookmeyer, Ron; Eshleman, Susan H.

2014-01-01

Background Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. Methods Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. Results The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. Conclusions MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation. PMID:24968135

Analysis of HIV Diversity in HIV-Infected Black Men Who Have Sex with Men (HPTN 061)

PubMed Central

Chen, Iris; Chau, Gordon; Wang, Jing; Clarke, William; Marzinke, Mark A.; Cummings, Vanessa; Breaud, Autumn; Laeyendecker, Oliver; Fields, Sheldon D.; Griffith, Sam; Scott, Hyman M.; Shoptaw, Steven; del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P.; Mayer, Kenneth H.; Koblin, Beryl A.; Eshleman, Susan H.

2016-01-01

Background HIV populations often diversify in response to selective pressures, such as the immune response and antiretroviral drug use. We analyzed HIV diversity in Black men who have sex with men who were enrolled in the HIV Prevention Trials Network 061 study. Methods A high resolution melting (HRM) diversity assay was used to measure diversity in six regions of the HIV genome: two in gag, one in pol, and three in env. HIV diversity was analyzed for 146 men who were HIV infected at study enrollment, including three with acute infection and 13 with recent infection (identified using a multi-assay algorithm), and for 21 men who seroconverted during the study. HIV diversification was analyzed in a paired analysis for 62 HIV-infected men using plasma samples from the enrollment and 12-month (end of study) visits. Results Men with acute or recent infection at enrollment and seroconverters had lower median HRM scores (lower HIV diversity) than men with non-recent infection in all six regions analyzed. In univariate analyses, younger age, higher CD4 cell count, and HIV drug resistance were associated with lower median HRM scores in multiple regions; ARV drug detection was marginally associated with lower diversity in the pol region. In multivariate analysis, acute or recent infection (all six regions) and HIV drug resistance (both gag regions) were associated with lower median HRM scores. Diversification in the pol region over 12 months was greater for men with acute or recent infection, higher CD4 cell count, and lower HIV viral load at study enrollment. Conclusions HIV diversity was significantly associated with duration of HIV infection, and lower gag diversity was observed in men who had HIV drug resistance. HIV pol diversification was more pronounced in men with acute or recent infection, higher CD4 cell count, and lower HIV viral load. PMID:27936098

Identification of a new genetic marker in Mycoplasma synoviae vaccine strain MS-H and development of a strategy using polymerase chain reaction and high-resolution melting curve analysis for differentiating MS-H from field strains.

PubMed

Zhu, Ling; Konsak, Barbara M; Olaogun, Olusola M; Agnew-Crumptona, Rebecca; Kanci, Anna; Marenda, Marc S; Browning, Glenn F; Noormohammadi, Amir H

2017-10-01

Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts + ) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic Pathways and Networks Associated With Tobacco Use in Military Personnel.

PubMed

Jones, Dean P; Walker, Douglas I; Uppal, Karan; Rohrbeck, Patricia; Mallon, Col Timothy M; Go, Young-Mi

2016-08-01

The aim of this study is to use high-resolution metabolomics (HRM) to identify metabolic pathways and networks associated with tobacco use in military personnel. Four hundred deidentified samples obtained from the Department of Defense Serum Repository were classified as tobacco users or nonusers according to cotinine content. HRM and bioinformatic methods were used to determine pathways and networks associated with classification. Eighty individuals were classified as tobacco users compared with 320 nonusers on the basis of cotinine levels at least 10 ng/mL. Alterations in lipid and xenobiotic metabolism, and diverse effects on amino acid, sialic acid, and purine and pyrimidine metabolism were observed. Importantly, network analysis showed broad effects on metabolic associations not simply linked to well-defined pathways. Tobacco use has complex metabolic effects that must be considered in evaluation of deployment-associated environmental exposures in military personnel.

Teaching and Learning about Managing People: Human Resource Management for Education Settings--A Queensland Case.

ERIC Educational Resources Information Center

McCrea, Nadine L.; Ehrich, Lisa C.

1996-01-01

Describes the development of a human resource management (HRM) elective for educators aspiring to management positions in various educational settings. The unit is part of a graduate diploma offered within the Faculty of Education at one Australian university. The paper argues the necessity of HRM studies in postgraduate education courses. (SM)

Standards in Action. Using National Standards for Human Resource Management and Development in Further Education. 1. Laying the Foundations.

ERIC Educational Resources Information Center

Further Education Unit, London (England).

This booklet is designed to assist British further education college managers responsible for quality assurance, human resources management (HRM), human resources development (HRD), corporate development, strategic planning, staffing/personnel, and curriculum in using national standards for HRM and HRD in further education. A synopsis, users'…

Finding the "Right Staff" in Small Firms

ERIC Educational Resources Information Center

Barrett, Rowena; Neeson, Robyn; Billington, Leo

2007-01-01

Purpose: The purpose of this paper is to explore what owners of small firms are looking for from new employees. The aim is to pursue this in light of the debate around formality and informality of small firm HRM, exploring the extent to which the small firms studied had formalized HRM practices. Design/methodology/approach: The data reported here…

A modified homogeneous relaxation model for CO2 two-phase flow in vapour ejector

NASA Astrophysics Data System (ADS)

Haida, M.; Palacz, M.; Smolka, J.; Nowak, A. J.; Hafner, A.; Banasiak, K.

2016-09-01

In this study, the homogenous relaxation model (HRM) for CO2 flow in a two-phase ejector was modified in order to increase the accuracy of the numerical simulations The two- phase flow model was implemented on the effective computational tool called ejectorPL for fully automated and systematic computations of various ejector shapes and operating conditions. The modification of the HRM was performed by a change of the relaxation time and the constants included in the relaxation time equation based on the experimental result under the operating conditions typical for the supermarket refrigeration system. The modified HRM was compared to the HEM results, which were performed based on the comparison of motive nozzle and suction nozzle mass flow rates.

Miniature Biometric Sensor Project

NASA Technical Reports Server (NTRS)

Falker, John; Terrier, Douglas; Clayton, Ronald; Hanson, Andrea; Cooper, Tommy; Downs, Meghan; Flint, Stephanie; Reyna, Baraquiel; Simon, Cory; Wilt, Grier

2015-01-01

Heart rate monitoring (HRM) is a critical need during exploration missions. Unlike the four separate systems used on ISS today, the single HRM system should perform as a diagnostic tool, perform well during exercise or high level activity, and be suitable for use during EVA. Currently available HRM technologies are dependent on uninterrupted contact with the skin and are prone to data drop-out and motion artifact when worn in the spacesuit or during exercise. Here, we seek an alternative to the chest strap and electrode based sensors currently in use on ISS today. This project aims to develop a single, high performance, robust biosensor with focused efforts on improved heart rate data quality collection during high intensity activity such as exercise or EVA.

Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E.

PubMed

Maurischat, Sven; Szabo, Istvan; Baumann, Beatrice; Malorny, Burkhard

2015-05-01

Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time. Copyright © 2015 Elsevier B.V. All rights reserved.

Pan-sharpening via compressed superresolution reconstruction and multidictionary learning

NASA Astrophysics Data System (ADS)

Shi, Cheng; Liu, Fang; Li, Lingling; Jiao, Licheng; Hao, Hongxia; Shang, Ronghua; Li, Yangyang

2018-01-01

In recent compressed sensing (CS)-based pan-sharpening algorithms, pan-sharpening performance is affected by two key problems. One is that there are always errors between the high-resolution panchromatic (HRP) image and the linear weighted high-resolution multispectral (HRM) image, resulting in spatial and spectral information lost. The other is that the dictionary construction process depends on the nontruth training samples. These problems have limited applications to CS-based pan-sharpening algorithm. To solve these two problems, we propose a pan-sharpening algorithm via compressed superresolution reconstruction and multidictionary learning. Through a two-stage implementation, compressed superresolution reconstruction model reduces the error effectively between the HRP and the linear weighted HRM images. Meanwhile, the multidictionary with ridgelet and curvelet is learned for both the two stages in the superresolution reconstruction process. Since ridgelet and curvelet can better capture the structure and directional characteristics, a better reconstruction result can be obtained. Experiments are done on the QuickBird and IKONOS satellites images. The results indicate that the proposed algorithm is competitive compared with the recent CS-based pan-sharpening methods and other well-known methods.

Bluetooth(Registered Trademark) Heart Rate Monitors for Spaceflight

NASA Technical Reports Server (NTRS)

Buxton, Roxanne E.; West, Michael R.; Kalogera, Kent L.; Hanson, Andrea M.

2016-01-01

Heart rate monitoring is required during exercise for crewmembers aboard the International Space Station (ISS) and will be for future exploration missions. The cardiovascular system must be sufficiently stressed throughout a mission to maintain the ability to perform nominal and contingency/emergency tasks. High quality heart rate data is required to accurately determine the intensity of exercise performed by the crewmembers and show maintenance of VO2max. The quality of the data collected on ISS is subject to multiple limitations and is insufficient to meet current requirements. PURPOSE: To evaluate the performance of commercially available Bluetooth® heart rate monitors (BT_HRM) and their ability to provide high quality heart rate data to monitor crew health on board ISS and during future exploration missions. METHODS: Nineteen subjects completed 30 data collection sessions of various intensities on the treadmill and/or cycle. Subjects wore several BT_HRM technologies for each testing session. One electrode-based chest strap (CS) was worn, while one or more optical sensors (OS) was worn. Subjects were instrumented with a 12-lead ECG to compare the heart rate data from the Bluetooth sensors. Each BT_RHM data set was time matched to the ECG data and a +/-5bpm threshold was applied to the difference between the two data sets. Percent error was calculated based on the number of data points outside the threshold and the total number of data points. REULTS: The electrode-based chest straps performed better than the optical sensors. The best performing CS was CS1 (1.6%error), followed by CS4 (3.3%error), CS3 (6.4%error), and CS2 (9.2%error). The OS resulted in 10.4% error for OS1 and 14.9% error for OS2. CONCLUSIONS: The highest quality data came from CS1, unfortunately it has been discontinued by the manufacturer. The optical sensors have not been ruled out for use, but more investigation is needed to determine how to get the best quality data. CS2 will be used in an ISS Bluetooth validation study, because it simultaneously transmits Magnetic Pulse which is integrated with existing exercise hardware on ISS. The simultaneous data streams allow for beat to beat comparison between the current ISS standard and CS2.Upon Bluetooth(Registered Trademark) validation aboard ISS, down select of a new BT_HRM for operational use will be made.

Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

PubMed

Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2013-01-01

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

PubMed Central

Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2013-01-01

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM. PMID:23620739

[Polar S810 as an alternative resource to the use of the electrocardiogram in the 4-second exercise test].

PubMed

Pimentel, Alan Santos; Alves, Eduardo da Silva; Alvim, Rafael de Oliveira; Nunes, Rogério Tasca; Costa, Carlos Magno Amaral; Lovisi, Júlio Cesar Moraes; Perrout de Lima, Jorge Roberto

2010-05-01

The 4-second exercise test (T4s) evaluates the cardiac vagal tone during the initial heart rate (HR) transient at sudden dynamic exercise, through the identification of the cardiac vagal index (CVI) obtained from the electrocardiogram (ECG). To evaluate the use of the Polar S810 heart rate monitor (HRM) as an alternative resource to the use of the electrocardiogram in the 4-second exercise test. In this study, 49 male individuals (25 +/- 20 years, 176 +/-12 cm, 74 +/- 6 kg) underwent the 4-second exercise test. The RR intervals were recorded simultaneously by ECG and HRM. We calculated the mean and the standard deviation of the last RR interval of the pre-exercise period, or of the first RR interval of the exercise period, whichever was longer (RRB), of the shortest RR interval of the exercise period (RRC), and of the CVI obtained by ECG and HRM. We used the Student t-test for dependent samples (p < or 0.05) to test the significance of the differences between means. To identify the correlation between the ECG and the HRM, we used the linear regression to calculate the Pearson's correlation coefficient and the strategy proposed by Bland and Altman. Linear regression showed r(2) of 0.9999 for RRB, 0.9997 for RRC, and 0.9996 for CVI. Bland e Altman strategy presented standard deviation of 0.92 ms for RRB, 0.86 ms for RRC, and 0.002 for CVI. Polar S810 HRM was more efficient in the application of T4s compared to the ECG.

The Professionalization of Human Resource Management: Examining Undergraduate Curricula and the Influence of Professional Organizations

ERIC Educational Resources Information Center

Parks-Leduc, Laura; Rutherford, Matthew A.; Becker, Karen L.; Shahzad, Ali M.

2018-01-01

This study explores the state of undergraduate human resource management (HRM) curricula worldwide in an effort to understand the extent to which there is an agreed-upon body of knowledge underpinning the field of HRM. We reviewed the undergraduate curricula for all business schools that were accredited by either the Association to Advance…

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

PubMed

Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

2015-12-01

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. Copyright © 2015 Elsevier Inc. All rights reserved.

Hard QCD rescattering in few nucleon systems

NASA Astrophysics Data System (ADS)

Maheswari, Dhiraj; Sargsian, Misak

2017-01-01

The theoretical framework of hard QCD rescattering mechanism (HRM) is extended to calculate the high energy γ3 He -> pd reaction at 900 center of mass angle. In HRM model , the incoming high energy photon strikes a quark from one of the nucleons in the target which subsequently undergoes hard rescattering with the quarks from the other nucleons generating hard two-body baryonic system in the final state of the reaction. Based on the HRM, a parameter free expression for the differential cross section for the reaction is derived, expressed through the 3 He -> pd transition spectral function, hard pd -> pd elastic scattering cross section and the effective charge of the quarks being interchanged in the hard rescattering process. The numerical estimates obtained from this expression for the differential cross section are in a good agreement with the data recently obtained at the Jefferson Lab experiment, showing the energy scaling of cross section with an exponent of s-17, also consistent with the quark counting rule. The angular and energy dependences of the cross section are also predicted within HRM which are in good agreement with the preliminary data of these distributions. Research is supported by the US Department of Energy.

Is there a role for high resolution manometry in GERD diagnosis?

PubMed

Tolone, Salvatore; Savarino, Edoardo; Docimo, Ludovico

2017-09-01

Gastroesophageal reflux disease (GERD) is a condition, which develops when the reflux of stomach contents causes troublesome symptoms and/or complications. In patients with alarm symptoms or signs or in those refractory to antiacid therapy, it is indicated to perform instrumental tests to correctly diagnose GERD presence. Esophageal manometry is currently considered the gold standard test for the diagnosis of esophageal dysmotility. However, it has shown limited capability in diagnosing GERD. With the advent of high resolution manometry (HRM), more accurate evaluations of esophageal motility are now possible. Furthermore, new metrics have been developed to investigate esophagogastric junction (EGJ) morphology and function. In particular, the antireflux barrier function of EGJ can now be assessed evaluating the contraction integral of the junction. Also, transient lower esophageal relaxations can be defined more precisely with HRM. Provocative tests with multiple swallows can give additional information on peristaltic reserve. Finally, adding impedance measurement to HRM allows to detect retrograde movements (e.g. reflux) in the esophagus and to measure the baseline impedance, a novel promising metric associated to esophageal mucosal integrity. The present review will discuss the recent progresses made in evaluation of GERD patients with HRM and its potential role in diagnosing pathological reflux presence.

Loss of Peristaltic Reserve, Determined by Multiple Rapid Swallows, Is the Most Frequent Esophageal Motility Abnormality in Patients With Systemic Sclerosis.

PubMed

Carlson, Dustin A; Crowell, Michael D; Kimmel, Jessica N; Patel, Amit; Gyawali, C Prakash; Hinchcliff, Monique; Griffing, W Leroy; Pandolfino, John E; Vela, Marcelo F

2016-10-01

We assessed peristaltic reserve using multiple rapid swallows (MRS) during esophageal high-resolution manometry (HRM) of 111 patients with systemic sclerosis (89 women; ages, 42-64 y). We performed a retrospective analysis of HRM studies that included MRS in patients with systemic sclerosis, performed at 2 tertiary referral centers, and compared data with those from 18 healthy volunteers (controls). HRM findings were analyzed according to the Chicago Classification to provide an esophageal motility diagnosis. Response to MRS was evaluated for the presence of contraction and for augmentation, defined as the distal contractile integral after MRS greater than the median distal contractile integral of 10 supine swallows. Esophageal motility diagnoses included 41% with absent contractility, 31% with normal motility, 23% with ineffective esophageal motility, and 5% that met the criteria for other esophageal motility disorders. Contraction (37%) and peristaltic augmentation (18%) after MRS were observed less frequently in patients with systemic sclerosis than in controls (83% and 100%, respectively). Impaired peristaltic reserve, as assessed with MRS during HRM, is therefore the most common esophageal motility finding among patients with systemic sclerosis. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Effects of Head Rotation and Head Tilt on Pharyngeal Pressure Events Using High Resolution Manometry

PubMed Central

Kim, Cheol Ki; Song, Sun Hong; Koo, Jung Hoi; Lee, Kyung Duck; Park, Hee Sun; Oh, Yoongul; Min, Kyunghoon

2015-01-01

Objective To observe changes in pharyngeal pressure during the swallowing process according to postures in normal individuals using high-resolution manometry (HRM). Methods Ten healthy volunteers drank 5 mL of water twice while sitting in a neutral posture. Thereafter, they drank the same amount of water twice in the head rotation and head tilting postures. The pressure and time during the deglutition process for each posture were measured with HRM. The data obtained for these two postures were compared with those obtained from the neutral posture. Results The maximum pressure, area, rise time, and duration in velopharynx (VP) and tongue base (TB) were not affected by changes in posture. In comparison, the maximum pressure and the pre-upper esophageal sphincter (UES) maximum pressure of the lower pharynx in the counter-catheter head rotation posture were lower than those in the neutral posture. The lower pharynx pressure in the catheter head tilting posture was higher than that in the counter-catheter head tilting. The changes in the VP peak and epiglottis, VP and TB peaks, and the VP onset and post-UES time intervals were significant in head tilting and head rotation toward the catheter postures, as compared with neutral posture. Conclusion The pharyngeal pressure and time parameter analysis using HRM determined the availability of head rotation as a compensatory technique for safe swallowing. Tilting the head smoothes the progress of food by increasing the pressure in the pharynx. PMID:26161349

Utility of Squeeze Flow in the Food Industry

NASA Astrophysics Data System (ADS)

Huang, T. A.

2008-07-01

Squeeze flow for obtaining shear viscosity on Newtonian and non-Newtonian fluids has long been established in the literature. Rotational shear flow using cone/plate, a set of parallel plates, or concentric cylinders all develop wall slip, shear fracture, or instability on food related materials such as peanut butter or mayonnaise. Viscosity data obtained using any one of the above mentioned set-ups is suspect or potentially results in significant error. They are unreliable to support or predict the textural differences perceived by consumer evaluation. RMS-800, from Rheometrics Inc., was employed to conduct the squeezing flow under constant speeds on a set of parallel plates. Viscosity data, over a broad range of shear rates, is compared between Hellmann's real (HRM) and light mayonnaise (HLM). The Consistency and shear-thinning indices, as defined in the Power-Law Model, were determined. HRM exhibits a more pronounced shear-thinning when compared to HLM yet the Consistency of HRM is significantly higher. Sensory evaluation by a trained expert panel ranked that adhesiveness and cohesiveness of HLM are significantly higher. It appears that the degree of shear thinning is one of the key rheological parameters in predicting the above mentioned difference in textural attributes. Error involved in determining viscosity from non-parallelism between two plates can be significant to affect the accuracy of the viscosity, in particular, shear-thinning index. Details are a subject for the next presentation. Nevertheless, the method is proven to be fast, rugged, simple, and reliable. It can be developed as a QC tool.

Esophageal motor function: technical aspects of manometry.

PubMed

Gyawali, C Prakash; Patel, Amit

2014-10-01

High-resolution manometry (HRM) has advanced the understanding of esophageal peristaltic mechanisms and has simplified esophageal motor testing. In this article the technical aspects of HRM are addressed, focusing on test protocols, in addition to concerns and pitfalls in performing esophageal motor studies. Specifically, catheter positioning, equipment-related artifacts, basal data acquisition, adequate swallows, and provocative maneuvers are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Intuitive Speech-based Robotic Control

DTIC Science & Technology

2010-04-01

NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Research Laboratory ATTN: RDRL- HRM -DW Aberdeen Proving Ground, MD 21005-5425 8...controlling helicopters, executing telephone and automobile functions, automating the handling of customer calls, converting spoken language into sign...RESEARCH LAB RDRL CIM P 2800 POWDER MILL RD ADELPHI MD 20783-1197 1 ARMY RSCH LABORATORY – HRED RDRL HRM A J MARTIN MYER CENTER BLDG

The impact of union elections on human resources management practices in hospitals.

PubMed

Deshpande, Satish P

2002-06-01

The purpose of this article is to explore top management's perceptions of how various human resources management (HRM) practices changed in hospitals (n = 101) after union elections. Significant increases in many HRM practices that are believed to lead to competitive advantage through human resources were reported in firms in which unions lost elections but not in firms where unions were certified.

Projecting Individualized Absolute Invasive Breast Cancer Risk in US Hispanic Women.

PubMed

Banegas, Matthew P; John, Esther M; Slattery, Martha L; Gomez, Scarlett Lin; Yu, Mandi; LaCroix, Andrea Z; Pee, David; Chlebowski, Rowan T; Hines, Lisa M; Thompson, Cynthia A; Gail, Mitchell H

2017-02-01

There is no model to estimate absolute invasive breast cancer risk for Hispanic women. The San Francisco Bay Area Breast Cancer Study (SFBCS) provided data on Hispanic breast cancer case patients (533 US-born, 553 foreign-born) and control participants (464 US-born, 947 foreign-born). These data yielded estimates of relative risk (RR) and attributable risk (AR) separately for US-born and foreign-born women. Nativity-specific absolute risks were estimated by combining RR and AR information with nativity-specific invasive breast cancer incidence and competing mortality rates from the California Cancer Registry and Surveillance, Epidemiology, and End Results program to develop the Hispanic risk model (HRM). In independent data, we assessed model calibration through observed/expected (O/E) ratios, and we estimated discriminatory accuracy with the area under the receiver operating characteristic curve (AUC) statistic. The US-born HRM included age at first full-term pregnancy, biopsy for benign breast disease, and family history of breast cancer; the foreign-born HRM also included age at menarche. The HRM estimated lower risks than the National Cancer Institute's Breast Cancer Risk Assessment Tool (BCRAT) for US-born Hispanic women, but higher risks in foreign-born women. In independent data from the Women's Health Initiative, the HRM was well calibrated for US-born women (observed/expected [O/E] ratio = 1.07, 95% confidence interval [CI] = 0.81 to 1.40), but seemed to overestimate risk in foreign-born women (O/E ratio = 0.66, 95% CI = 0.41 to 1.07). The AUC was 0.564 (95% CI = 0.485 to 0.644) for US-born and 0.625 (95% CI = 0.487 to 0.764) for foreign-born women. The HRM is the first absolute risk model that is based entirely on data specific to Hispanic women by nativity. Further studies in Hispanic women are warranted to evaluate its validity. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.

Human resources for health policies: a critical component in health policies

PubMed Central

Dussault, Gilles; Dubois, Carl-Ardy

2003-01-01

In the last few years, increasing attention has been paid to the development of health policies. But side by side with the presumed benefits of policy, many analysts share the opinion that a major drawback of health policies is their failure to make room for issues of human resources. Current approaches in human resources suggest a number of weaknesses: a reactive, ad hoc attitude towards problems of human resources; dispersal of accountability within human resources management (HRM); a limited notion of personnel administration that fails to encompass all aspects of HRM; and finally the short-term perspective of HRM. There are three broad arguments for modernizing the ways in which human resources for health are managed: • the central role of the workforce in the health sector; • the various challenges thrown up by health system reforms; • the need to anticipate the effect on the health workforce (and consequently on service provision) arising from various macroscopic social trends impinging on health systems. The absence of appropriate human resources policies is responsible, in many countries, for a chronic imbalance with multifaceted effects on the health workforce: quantitative mismatch, qualitative disparity, unequal distribution and a lack of coordination between HRM actions and health policy needs. Four proposals have been put forward to modernize how the policy process is conducted in the development of human resources for health (HRH): • to move beyond the traditional approach of personnel administration to a more global concept of HRM; • to give more weight to the integrated, interdependent and systemic nature of the different components of HRM when preparing and implementing policy; • to foster a more proactive attitude among human resources (HR) policy-makers and managers; • to promote the full commitment of all professionals and sectors in all phases of the process. The development of explicit human resources policies is a crucial link in health policies and is needed both to address the imbalances of the health workforce and to foster implementation of the health services reforms. PMID:12904254

Human resources for health policies: a critical component in health policies.

PubMed

Dussault, Gilles; Dubois, Carl-Ardy

2003-04-14

In the last few years, increasing attention has been paid to the development of health policies. But side by side with the presumed benefits of policy, many analysts share the opinion that a major drawback of health policies is their failure to make room for issues of human resources. Current approaches in human resources suggest a number of weaknesses: a reactive, ad hoc attitude towards problems of human resources; dispersal of accountability within human resources management (HRM); a limited notion of personnel administration that fails to encompass all aspects of HRM; and finally the short-term perspective of HRM.There are three broad arguments for modernizing the ways in which human resources for health are managed:bullet; the central role of the workforce in the health sector;bullet; the various challenges thrown up by health system reforms;bullet; the need to anticipate the effect on the health workforce (and consequently on service provision) arising from various macroscopic social trends impinging on health systems.The absence of appropriate human resources policies is responsible, in many countries, for a chronic imbalance with multifaceted effects on the health workforce: quantitative mismatch, qualitative disparity, unequal distribution and a lack of coordination between HRM actions and health policy needs.Four proposals have been put forward to modernize how the policy process is conducted in the development of human resources for health (HRH):bullet; to move beyond the traditional approach of personnel administration to a more global concept of HRM;bullet; to give more weight to the integrated, interdependent and systemic nature of the different components of HRM when preparing and implementing policy;bullet; to foster a more proactive attitude among human resources (HR) policy-makers and managers;bullet; to promote the full commitment of all professionals and sectors in all phases of the process.The development of explicit human resources policies is a crucial link in health policies and is needed both to address the imbalances of the health workforce and to foster implementation of the health services reforms.

Anchoring Linkage Groups of the Rosa Genetic Map to Physical Chromosomes with Tyramide-FISH and EST-SNP Markers

PubMed Central

Kirov, Ilya; Van Laere, Katrijn; De Riek, Jan; De Keyser, Ellen; Van Roy, Nadine; Khrustaleva, Ludmila

2014-01-01

In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb–1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria. PMID:24755945

Naval Leadership: A Study of Views on Leadership Competencies and Methods to Reinforce Leadership Skills

DTIC Science & Technology

1990-12-01

stated these problems -- racism , sexism , drug and alcohol abuse -- were a result of the poor leadership ability in Navymiddle management [Ref. 3... The HRM program instituted a formal course of instruction to teach leadership theories . The leadership training of the Human Resource Management...management practices based on the guidelines developed by W. E. Demming [Ref. 14]. The TQL practice involves integrating management and statistical methods to

Interaction of reelin and stress on immobility in the forced swim test but not dopamine-mediated locomotor hyperactivity or prepulse inhibition disruption: Relevance to psychotic and mood disorders.

PubMed

Notaras, Michael J; Vivian, Billie; Wilson, Carey; van den Buuse, Maarten

2017-07-13

Psychotic disorders, such as schizophrenia, as well as some mood disorders, such as bipolar disorder, have been suggested to share common biological risk factors. One such factor is reelin, a large extracellular matrix glycoprotein that regulates neuronal migration during development as well as numerous activity-dependent processes in the adult brain. The current study sought to evaluate whether a history of stress exposure interacts with endogenous reelin levels to modify behavioural endophenotypes of relevance to psychotic and mood disorders. Heterozygous Reeler Mice (HRM) and wildtype (WT) controls were treated with 50mg/L of corticosterone (CORT) in their drinking water from 6 to 9weeks of age, before undergoing behavioural testing in adulthood. We assessed methamphetamine-induced locomotor hyperactivity, prepulse inhibition (PPI) of acoustic startle, short-term spatial memory in the Y-maze, and depression-like behaviour in the Forced-Swim Test (FST). HRM genotype or CORT treatment did not affect methamphetamine-induced locomotor hyperactivity, a model of psychosis-like behaviour. At baseline, HRM showed decreased PPI at the commonly used 100msec interstimulus interval (ISI), but not at the 30msec ISI or following challenge with apomorphine. A history of CORT exposure potentiated immobility in the FST amongst HRM, but not WT mice. In the Y-maze, chronic CORT treatment decreased novel arm preference amongst HRM, reflecting reduced short-term spatial memory. These data confirm a significant role of endogenous reelin levels on stress-related behaviour, supporting a possible role in both bipolar disorder and schizophrenia. However, an interaction of reelin deficiency with dopaminergic regulation of psychosis-like behaviour remains unclear. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of H2O2 stress-responsive genes through a novel transcription factor in the protozoan pathogen Entamoeba histolytica.

PubMed

Pearson, Richard J; Morf, Laura; Singh, Upinder

2013-02-08

Outcome of infection depends upon complex interactions between the invading pathogen and the host. As part of the host's innate immune response, the release of reactive oxygen and nitrogen species by phagocytes represents a major obstacle to the establishment of infection. The ability of the human parasite Entamoeba histolytica to survive reactive oxygen and nitrogen species is central to its pathogenic potential and contributes to disease outcome. In order to define the transcriptional network associated with oxidative stress, we utilized the MEME and MAST programs to analyze the promoter regions of 57 amoebic genes that had increased expression specifically in response to H(2)O(2) exposure. We functionally characterized an H(2)O(2)-regulatory motif (HRM) ((1)AAACCTCAATGAAGA(15)), which was enriched in these promoters and specifically bound amoebic nuclear protein(s). Assays with promoter-luciferase fusions established the importance of key residues and that the HRM motif directly impacted the ability of H(2)O(2)-responsive promoters to drive gene expression. DNA affinity chromatography and mass spectrometry identified EHI_108720 as an HRM DNA-binding protein. Overexpression and down-regulation of EHI_108720 demonstrated the specificity of EHI_108720 protein binding to the HRM, and overexpression increased basal expression from an H(2)O(2)-responsive wild-type promoter but not from its mutant counterpart. Thus, EHI_108720, or HRM-binding protein, represents a new stress-responsive transcription factor in E. histolytica that controls a transcriptional regulatory network associated with oxidative stress. Overexpression of EHI_108720 increased parasite virulence. Insight into how E. histolytica responds to oxidative stress increases our understanding of how this important human pathogen establishes invasive disease.

Clinicomanometric factors associated with clinically relevant esophagogastric junction outflow obstruction from the Sandhill high-resolution manometry system.

PubMed

Song, B G; Min, Y W; Lee, H; Min, B-H; Lee, J H; Rhee, P-L; Kim, J J

2018-03-01

Integrated relaxation pressure (IRP) is a key metric for diagnosing esophagogastric junction outflow obstruction (EGJOO). However, its normal value might be different according to the manufacturer of high-resolution manometry (HRM). This study aimed to investigate optimal value of IRP for diagnosing EGJOO in Sandhill HRM and to find clinicomanometric variables to segregate clinically relevant EGJOO. We analyzed 262 consecutive subjects who underwent HRM between June 2011 and December 2016 showing elevated median IRP (> 15 mm Hg) but did not satisfy criteria for achalasia. Clinically relevant subjects were defined as follows: (i) subsequent HRM met achalasia criteria during follow-up (early achalasia); (ii) Eckardt score was decreased at least two points without exceeding a score of 3 after pneumatic dilatation (variant achalasia); and (iii) significant passage disturbance on esophagogram without structural abnormality (possible achalasia). Seven subjects were clinically relevant, including two subjects with early achalasia, four subjects with variant achalasia, and one subject with possible achalasia. All clinically relevant subjects had IRP 20 mm Hg or above. Among subjects (n = 122) with IRP 20 mm Hg or more, clinically relevant group (n = 7) had significantly higher rate of dysphagia (100% vs 24.3%, P < .001) and compartmentalized pressurization (85.7% vs 21.7%, P = .001) compared to clinically non-relevant group (n = 115). Our results suggest that IRP of 20 mm Hg or higher could segregate clinically relevant subjects showing EGJOO in Sandhill HRM. Additionally, if subjects have both dysphagia and compartmentalized pressurization, careful follow-up is essential. © 2017 John Wiley & Sons Ltd.

Predictive value of routine esophageal high-resolution manometry for gastro-esophageal reflux disease.

PubMed

van Hoeij, F B; Smout, A J; Bredenoord, A J

2015-07-01

Using conventional manometry, gastro-esophageal reflux disease (GERD) was associated with a reduced lower esophageal sphincter (LES) pressure and impaired peristalsis. However, with a large overlap between GERD patients and controls, these findings are of limited clinical relevance. It is uncertain whether the more detailed information of high-resolution manometry (HRM) can discriminate GERD patients. Therefore, we aimed to determine to which extent HRM findings can predict GERD. HRM measurements in 69 patients with GERD and 40 healthy subjects were compared and the predictive value of HRM for the diagnosis of GERD was explored. GERD patients had a significantly lower contraction amplitude (55 vs 64 mmHg; p = 0.045) and basal LES pressure (10 vs 13.2 mmHg; p = 0.034) than healthy controls. GERD patients more often had a hiatal hernia than healthy subjects (30% vs 7%; p = 0.005). Patients with reflux esophagitis had a lower DCI than patients without reflux esophagitis (558 vs 782 mmHg cm s; p = 0.045). No significant difference was seen in contractile front velocity, distal latency, number of peristaltic breaks, residual LES pressure and LES length. On multivariate logistic regression analysis, both esophagogastric junction type I (OR 4.971; 95% CI 1.33-18.59; p = 0.017) and mean wave amplitude (OR 0.95; 95% CI 0.90-0.98; p = 0.013) were found to be independent predictors of GERD. However, the sensitivity and specificity of these findings were low. Hiatal hernia, low contraction amplitude and LES pressure are associated with GERD, but do not predict the disease with sufficient accuracy. Routine esophageal HRM can therefore not be used to distinguish GERD patients from healthy subjects. © 2015 John Wiley & Sons Ltd.

Gastroesophageal flap valve reflected EGJ morphology and correlated to acid reflux.

PubMed

Xie, Chenxi; Li, Yuwen; Zhang, Ning; Xiong, Lishou; Chen, Minhu; Xiao, Yinglian

2017-11-22

The anatomy of esophagogastric junction (EGJ) serves as the anti-reflux barrier. The gastroesophageal flap valve (GEFV) is a component of EGJ. The aim of the current study was to assess its correlation with the esophageal acid exposure and the impact on anti-reflux barrier function by using the metrics of EGJ contraction. Eighty three patients with typical GERD symptoms were included in the study. Upper endoscopy, high-resolution manometry (HRM) and 24 h multichannel intraluminal impedance-pH (MII-pH) monitoring were performed in all patients. GEFV was determined as four grades during endoscopic examination based on the Hill classification. The esophageal pressure topography (EPT) metrics defined in the updated Chicago Classification were measured by HRM, including integrated relaxation pressure (IRP), EGJ contractile index (EGJ-CI),expiratory EGJ pressure(EGJP-exp) and inspiratory EGJ pressure (EGJP-insp). The GEFV grade III and IV was more commonly found in patients with esophagitits (p < 0.05). The acid exposure time (AET%) and supine AET% were lower in patients with GEFV grade I (p < 0.01). There was weak correlation between AET% and GEFV grades (r = 0.27, p = 0.013). There were more EGJ morphology type III in patients with GEFV grade IV (p < 0.05).There were no significant differences on the values of four HRM metrics among the patients with different GEFV grades (p > 0.05). The GEFV grades were associated with acid reflux positively and could be a good reflection of EGJ morphology in HRM. But it had no impact on the four HRM metrics. Our research revealed that GEFV may play an assistant role in the anti-reflux barrier.

Inter-observer agreement for diagnostic classification of esophageal motility disorders defined in high-resolution manometry.

PubMed

Fox, M R; Pandolfino, J E; Sweis, R; Sauter, M; Abreu Y Abreu, A T; Anggiansah, A; Bogte, A; Bredenoord, A J; Dengler, W; Elvevi, A; Fruehauf, H; Gellersen, S; Ghosh, S; Gyawali, C P; Heinrich, H; Hemmink, M; Jafari, J; Kaufman, E; Kessing, K; Kwiatek, M; Lubomyr, B; Banasiuk, M; Mion, F; Pérez-de-la-Serna, J; Remes-Troche, J M; Rohof, W; Roman, S; Ruiz-de-León, A; Tutuian, R; Uscinowicz, M; Valdovinos, M A; Vardar, R; Velosa, M; Waśko-Czopnik, D; Weijenborg, P; Wilshire, C; Wright, J; Zerbib, F; Menne, D

2015-01-01

High-resolution esophageal manometry (HRM) is a recent development used in the evaluation of esophageal function. Our aim was to assess the inter-observer agreement for diagnosis of esophageal motility disorders using this technology. Practitioners registered on the HRM Working Group website were invited to review and classify (i) 147 individual water swallows and (ii) 40 diagnostic studies comprising 10 swallows using a drop-down menu that followed the Chicago Classification system. Data were presented using a standardized format with pressure contours without a summary of HRM metrics. The sequence of swallows was fixed for each user but randomized between users to avoid sequence bias. Participants were blinded to other entries. (i) Individual swallows were assessed by 18 practitioners (13 institutions). Consensus agreement (≤ 2/18 dissenters) was present for most cases of normal peristalsis and achalasia but not for cases of peristaltic dysmotility. (ii) Diagnostic studies were assessed by 36 practitioners (28 institutions). Overall inter-observer agreement was 'moderate' (kappa 0.51) being 'substantial' (kappa > 0.7) for achalasia type I/II and no lower than 'fair-moderate' (kappa >0.34) for any diagnosis. Overall agreement was somewhat higher among those that had performed >400 studies (n = 9; kappa 0.55) and 'substantial' among experts involved in development of the Chicago Classification system (n = 4; kappa 0.66). This prospective, randomized, and blinded study reports an acceptable level of inter-observer agreement for HRM diagnoses across the full spectrum of esophageal motility disorders for a large group of clinicians working in a range of medical institutions. Suboptimal agreement for diagnosis of peristaltic motility disorders highlights contribution of objective HRM metrics. © 2014 International Society for Diseases of the Esophagus.

Optimizing the high-resolution manometry (HRM) study protocol.

PubMed

Patel, A; Ding, A; Mirza, F; Gyawali, C P

2015-02-01

Intolerance of the esophageal manometry catheter may prolong high-resolution manometry (HRM) studies and increase patient distress. We assessed the impact of obtaining the landmark phase at the end of the study when the patient has acclimatized to the HRM catheter. 366 patients (mean age 55.4 ± 0.8 years, 62.0% female) undergoing esophageal HRM over a 1-year period were studied. The standard protocol consisted of the landmark phase, 10 5 mL water swallows 20-30 s apart, and multiple rapid swallows where 4-6 2 mL swallows were administered in rapid succession. The modified protocol consisted of the landmark phase at the end of the study after test swallows. Study duration, technical characteristics, indications, and motor findings were compared between standard and modified protocols. Of the 366 patients, 89.6% underwent the standard protocol (study duration 12.9 ± 0.3 min). In 10.4% with poor catheter tolerance undergoing the modified protocol, study duration was significantly longer (15.6 ± 1.0 min, p = 0.004) despite similar duration of study maneuvers. Only elevated upper esophageal sphincter basal pressures at the beginning of the study segregated modified protocol patients. The 95th percentile time to landmark phase in the standard protocol patients was 6.1 min; as many as 31.4% of modified protocol patients could not obtain their first study maneuver within this period (p = 0.0003). Interpretation was not impacted by shifting the landmark phase to the end of the study. Modification of the HRM study protocol with the landmark phase obtained at the end of the study optimizes study duration without compromising quality. © 2014 John Wiley & Sons Ltd.

Hard breakup of two nucleons from the He3 nucleus

NASA Astrophysics Data System (ADS)

Sargsian, Misak M.; Granados, Carlos

2009-07-01

We investigate a large angle photodisintegration of two nucleons from the He3 nucleus within the framework of the hard rescattering model (HRM). In the HRM a quark of one nucleon knocked out by an incoming photon rescatters with a quark of the other nucleon leading to the production of two nucleons with large relative momentum. Assuming the dominance of the quark-interchange mechanism in a hard nucleon-nucleon scattering, the HRM allows the expression of the amplitude of a two-nucleon breakup reaction through the convolution of photon-quark scattering, NN hard scattering amplitude, and nuclear spectral function, which can be calculated using a nonrelativistic He3 wave function. The photon-quark scattering amplitude can be explicitly calculated in the high energy regime, whereas for NN scattering one uses the fit of the available experimental data. The HRM predicts several specific features for the hard breakup reaction. First, the cross section will approximately scale as s-11. Second, the s11 weighted cross section will have the shape of energy dependence similar to that of s10 weighted NN elastic scattering cross section. Also one predicts an enhancement of the pp breakup relative to the pn breakup cross section as compared to the results from low energy kinematics. Another result is the prediction of different spectator momentum dependencies of pp and pn breakup cross sections. This is due to the fact that the same-helicity pp-component is strongly suppressed in the ground state wave function of He3. Because of this suppression the HRM predicts significantly different asymmetries for the cross section of polarization transfer NN breakup reactions for circularly polarized photons. For the pp breakup this asymmetry is predicted to be zero while for the pn it is close to (2)/(3).

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica*

PubMed Central

Pearson, Richard J.; Morf, Laura; Singh, Upinder

2013-01-01

Outcome of infection depends upon complex interactions between the invading pathogen and the host. As part of the host's innate immune response, the release of reactive oxygen and nitrogen species by phagocytes represents a major obstacle to the establishment of infection. The ability of the human parasite Entamoeba histolytica to survive reactive oxygen and nitrogen species is central to its pathogenic potential and contributes to disease outcome. In order to define the transcriptional network associated with oxidative stress, we utilized the MEME and MAST programs to analyze the promoter regions of 57 amoebic genes that had increased expression specifically in response to H2O2 exposure. We functionally characterized an H2O2-regulatory motif (HRM) (1AAACCTCAATGAAGA15), which was enriched in these promoters and specifically bound amoebic nuclear protein(s). Assays with promoter-luciferase fusions established the importance of key residues and that the HRM motif directly impacted the ability of H2O2-responsive promoters to drive gene expression. DNA affinity chromatography and mass spectrometry identified EHI_108720 as an HRM DNA-binding protein. Overexpression and down-regulation of EHI_108720 demonstrated the specificity of EHI_108720 protein binding to the HRM, and overexpression increased basal expression from an H2O2-responsive wild-type promoter but not from its mutant counterpart. Thus, EHI_108720, or HRM-binding protein, represents a new stress-responsive transcription factor in E. histolytica that controls a transcriptional regulatory network associated with oxidative stress. Overexpression of EHI_108720 increased parasite virulence. Insight into how E. histolytica responds to oxidative stress increases our understanding of how this important human pathogen establishes invasive disease. PMID:23250742

SYTO9 and SYBR GREEN1 with a high-resolution melting analysis for prenatal diagnosis of β⁰-thalassemia/hemoglobin-E.

PubMed

Pornprasert, Sakorn; Sukunthamala, Kanyakan

2010-11-01

The β⁰-thalassemia/Hb-E causes a wide range of severe conditions. A high medical cost is incurred in severe cases. Thus, the prevention of new cases of β⁰-thalassemia/Hb-E is required. The aim of this study is to use the SYTO9 and SYBR GREEN1 high-resolution melting (HRM) analysis for prenatal diagnosis of β⁰-thalassemia/Hb-E. DNA samples were extracted from amniotic fluid or cord blood of 11 pregnancies whose fetuses were at risk for β-thalassemia/Hb-E. PCR products from multiplex amplification refractory mutation system PCR for the detection of β⁰-thalassemia mutations at codons 17(A>T), 41/42(-TCTT), and 71/72(+A) and from amplification refractory mutation system PCR for the detection of Hb-E were characterized by SYTO9 HRM analysis. Moreover, β⁰-thalassemia 3.5- kb deletion was detected using real-time PCR with SYBR GREEN1 HRM analysis. Seven of 11 fetuses (64%) were diagnosed as β⁰-thalassemia/Hb-E (4 fetuses with mutation at codon 17, 2 with mutation at codon 41/42, and 1 with 3.5- kb deletion). Results from HRM analysis were completely consistent with those from fetal blood samplings analyzed at the time of delivery or pregnancy termination using HPLC. Therefore, the HRM analysis is easy to use. It is simple, flexible, non-destructive and has superb sensitivity and specificity. This approach might facilitate the laboratory diagnosis and genetic counseling for regions with a high prevalence of β⁰-thalassemia/Hb-E. © 2010 John Wiley & Sons A/S.

High Energy Break-Up of Few-Nucleon Systems

NASA Astrophysics Data System (ADS)

Sargsian, Misak

2008-03-01

We discus recent developments in theory of high energy two-body break-up reactions of few-nucleon systems. The characteristics of these reactions are such that the hard two-body quasielastic subprocess can be clearly separated from the accompanying soft subprocesses. We discuss in details the hard rescattering model (HRM) in which hard photodisintegration develops in two stages. At first, photon knocks-out an energetic quark which rescatters subsequently with a quark of the other nucleon. The latter provides a mechanism of sharing the initial high momentum of the photon by the outgoing two nucleons. Within HRM we discuss hard break-up reactions involving 2D and 3He targets. Another development of HRM is the prediction of new helicity selection mechanism for hard two-body reactions, which was apparently confirmed in the recent JLab experiment.

Analysis of SNP rs16754 of WT1 gene in a series of de novo acute myeloid leukemia patients.

PubMed

Luna, Irene; Such, Esperanza; Cervera, Jose; Barragán, Eva; Jiménez-Velasco, Antonio; Dolz, Sandra; Ibáñez, Mariam; Gómez-Seguí, Inés; López-Pavía, María; Llop, Marta; Fuster, Óscar; Oltra, Silvestre; Moscardó, Federico; Martínez-Cuadrón, David; Senent, M Leonor; Gascón, Adriana; Montesinos, Pau; Martín, Guillermo; Bolufer, Pascual; Sanz, Miguel A

2012-12-01

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

Communities of Practice: Towards Leveraging Knowledge in the Military

DTIC Science & Technology

2007-09-01

Sveiby (2001b) explains how the private sector went through a similar cycle in the 1980s that gave birth to the emergence of the modern “KM movement... 1980s (Svieby, 2001b) organizations were already quite proficient at “managing” knowledge. Consider the apprenticeship and mentorship programs...linking KM with HRM has 40 important implications for organizational performance. Hislop (2005) argues that Human Resource Management (HRM) practice

Flavonoid-Induced Expression of a Symbiosis-Related Gene in the Cyanobacterium Nostoc punctiforme

PubMed Central

Cohen, Michael F.; Yamasaki, Hideo

2000-01-01

The flavonoid naringin was found to induce the expression of hrmA, a gene with a symbiotic phenotype in the cyanobacterium Nostoc punctiforme. A comparative analysis of several flavonoids revealed the 7-O-neohesperidoside, 4′-OH, and C-2 000000000000 000000000000 000000000000 000000000000 111111111111 000000000000 000000000000 000000000000 000000000000 C-3 double bond in naringin as structural determinants of its hrmA-inducing activity. PMID:10913102

Life Stress and Coping Skills in Relation to Performance and Organizational Effectiveness.

DTIC Science & Technology

1980-05-15

have no such effect. Our work and the findings of others indicate that a build-up of negative events influences automobile accidents, morale on the Job...Research & Administrative Science Monterey, California 93940 LIST 7 HRM Officer in Charge Officer in Charge Human Resource Management Detachment Human...Pensacola, Florida 32508 Naval Training Equipment Center Orlando, Florida 32813 Naval Military Personnel Command (2 copies) HRM Department (NMPC-6

Validation of criteria for the definition of transient lower esophageal sphincter relaxations using high-resolution manometry.

PubMed

Roman, S; Holloway, R; Keller, J; Herbella, F; Zerbib, F; Xiao, Y; Bernard, L; Bredenoord, A J; Bruley des Varannes, S; Chen, M; Fox, M; Kahrilas, P J; Mittal, R K; Penagini, R; Savarino, E; Sifrim, D; Wu, J; Decullier, E; Pandolfino, J E; Mion, F

2017-02-01

Criteria for transient lower esophageal sphincter relaxations (TLESRs) are well-defined for Dentsleeve manometry. As high-resolution manometry (HRM) is now the gold standard to assess esophageal motility, our aim was to propose a consensus definition of TLESRs using HRM. Postprandial esophageal HRM combined with impedance was performed in 10 patients with gastroesophageal reflux disease. Transient lower esophageal sphincter relaxations identification was performed by 17 experts using a Delphi process. Four investigators then characterized TLESR candidates that achieved 100% agreement (TLESR events) and those that achieved less than 25% agreement (non-events) after the third round. Logistic regression and decision tree analysis were used to define optimal diagnostic criteria. All diagnostic criteria were more frequently encountered in the 57 TLESR events than in the 52 non-events. Crural diaphragm (CD) inhibition and LES relaxation duration >10 seconds had the highest predictive value to identify TLESR. Based on decision tree analysis, reflux on impedance, esophageal shortening, common cavity, upper esophageal sphincter relaxation without swallow and secondary peristalsis were alternate diagnostic criteria. Using HRM, TLESR might be defined as LES relaxation occurring in absence of swallowing, lasting more than 10 seconds and associated with CD inhibition. © 2016 John Wiley & Sons Ltd.

Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping

PubMed Central

Fraley, Stephanie I.; Athamanolap, Pornpat; Masek, Billie J.; Hardick, Justin; Carroll, Karen C.; Hsieh, Yu-Hsiang; Rothman, Richard E.; Gaydos, Charlotte A.; Wang, Tza-Huei; Yang, Samuel

2016-01-01

High Resolution Melt (HRM) is a versatile and rapid post-PCR DNA analysis technique primarily used to differentiate sequence variants among only a few short amplicons. We recently developed a one-vs-one support vector machine algorithm (OVO SVM) that enables the use of HRM for identifying numerous short amplicon sequences automatically and reliably. Herein, we set out to maximize the discriminating power of HRM + SVM for a single genetic locus by testing longer amplicons harboring significantly more sequence information. Using universal primers that amplify the hypervariable bacterial 16 S rRNA gene as a model system, we found that long amplicons yield more complex HRM curve shapes. We developed a novel nested OVO SVM approach to take advantage of this feature and achieved 100% accuracy in the identification of 37 clinically relevant bacteria in Leave-One-Out-Cross-Validation. A subset of organisms were independently tested. Those from pure culture were identified with high accuracy, while those tested directly from clinical blood bottles displayed more technical variability and reduced accuracy. Our findings demonstrate that long sequences can be accurately and automatically profiled by HRM with a novel nested SVM approach and suggest that clinical sample testing is feasible with further optimization. PMID:26778280

A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

PubMed

Menasherow, Sophia; Erster, Oran; Rubinstein-Giuni, Marisol; Kovtunenko, Anita; Eyngor, Evgeny; Gelman, Boris; Khinich, Evgeny; Stram, Yehuda

2016-06-01

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable. Copyright © 2016 Elsevier B.V. All rights reserved.

On Constructing, Grouping and Using Topical Ontology for Semantic Matching

NASA Astrophysics Data System (ADS)

Tang, Yan; de Baer, Peter; Zhao, Gang; Meersman, Robert

An ontology topic is used to group concepts from different contexts (or even from different domain ontologies). This paper presents a pattern-driven modeling methodology for constructing and grouping topics in an ontology (PAD-ON methodology), which is used for matching similarities between competences in the human resource management (HRM) domain. The methodology is supported by a tool called PAD-ON. This paper demonstrates our recent achievement in the work from the EC Prolix project. The paper approach is applied to the training processes at British Telecom as the test bed.

Characterization of fiber Bragg grating-based sensor array for high resolution manometry

NASA Astrophysics Data System (ADS)

Becker, Martin; Rothhardt, Manfred; Schröder, Kerstin; Voigt, Sebastian; Mehner, Jan; Teubner, Andreas; Lüpke, Thomas; Thieroff, Christoph; Krüger, Matthias; Chojetzki, Christoph; Bartelt, Hartmut

2012-04-01

The combination of fiber Bragg grating arrays integrated in a soft plastic tube is promising for high resolution manometry (HRM) where pressure measurements are done with high spatial resolution. The application as a medical device and in vivo experiments have to be anticipated by characterization with a measurement setup that simulates natural conditions. Good results are achieved with a pressure chamber which applies a well-defined pressure with a soft tubular membrane. It is shown that the proposed catheter design reaches accuracies down to 1 mbar and 1 cm.

High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

PubMed Central

Peña, Victor H.; Fernández, Geysson J.; Gómez-Palacio, Andrés M.; Mejía-Jaramillo, Ana M.; Cantillo, Omar; Triana-Chávez, Omar

2012-01-01

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate. PMID:22389739

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples.

PubMed

Masiero, Eva; Banik, Dipanwita; Abson, John; Greene, Paul; Slater, Adrian; Sgamma, Tiziana

2017-10-26

Liriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5' end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbcLa ) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs.

Detection of PIK3CA gene mutations with HRM analysis and association with IGFBP-5 expression levels in breast cancer.

PubMed

Dirican, Ebubekir; Kaya, Zehra; Gullu, Gokce; Peker, Irem; Ozmen, Tolga; Gulluoglu, Bahadir M; Kaya, Handan; Ozer, Ayse; Akkiprik, Mustafa

2014-01-01

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

Examining the Convergent and Discriminant Validity of Visual and Mental Workload Using Ocular Activity Variables

DTIC Science & Technology

2010-03-01

Laboratory ATTN: RDRL- HRM -DI Aberdeen Proving Ground, MD 21005-5425 8. PERFORMING ORGANIZATION REPORT NUMBER ARL-TR-5132 9. SPONSORING... Automobile drivers can successfully manage lateral movement and appropriate acceleration parameters, and listening to a radio does not appear to...JOHN J KINGMAN RD STE 0944 FT BELVOIR VA 22060-6218 1 ARL FIRES CENTER OF EXCELLENCE FIELD ELEMENT ATTN RDRL HRM AF C HERNANDEZ

Bibliography of Unclassified Reports: FY 1983.

DTIC Science & Technology

1983-11-01

selected automobile and motorcyclemagazines with a high proportion of readership in this age group. The strategy was evaluated by comparing the number of...climate of the human resource management ( HRM ) survey in compliance with the FY82 Navy Affirmative Action Plan. The most recent 1-year data base (I July...1980 to 30 June 1981) of HRM survey responses was used, yielding a sample of 35,690 personnel. The responses of whites and blacks, whites and

High-Resolution Metabolomics for Nutrition and Health Assessment of Armed Forces Personnel.

PubMed

Accardi, Carolyn Jonas; Walker, Douglas I; Uppal, Karan; Quyyumi, Arshed A; Rohrbeck, Patricia; Pennell, Kurt D; Mallon, Col Timothy M; Jones, Dean P

2016-08-01

The aim of this study was to test the utility of high-resolution metabolomics (HRM) for analysis of nutritional status and health indicators in military personnel. Serum samples from 400 military personnel were obtained from the Department of Defense Serum Repository (DoDSR) and analyzed for metabolites related to nutrition and health status. Metabolic profile organization was studied using modulated modularity clustering (MMC). HRM provided quantitative measures of 61 metabolites across chemical classes for use as nutritional and clinical biomarkers. Levels were comparable to reported values except for arginine and glutamine, which were above and below reference ranges, respectively. MMC generated five clusters, three of which were associated and contained amino acids. The others contained lipids and mitochondria-related metabolites. HRM analysis of serum is suitable for real-time and/or retrospective evaluation of nutrition and health status of specific military cohorts.

Influence of model grid size on the simulation of PM2.5 and the related excess mortality in Japan

NASA Astrophysics Data System (ADS)

Goto, D.; Ueda, K.; Ng, C. F.; Takami, A.; Ariga, T.; Matsuhashi, K.; Nakajima, T.

2016-12-01

Aerosols, especially PM2.5, can affect air pollution, climate change, and human health. The estimation of health impacts due to PM2.5 is often performed using global and regional aerosol transport models with various horizontal resolutions. To investigate the dependence of the simulated PM2.5 on model grid sizes, we executed two simulations using a high-resolution model ( 10km; HRM) and a low-resolution model ( 100km; LRM, which is a typical value for general circulation models). In this study, we used a global-to-regional atmospheric transport model to simulate PM2.5 in Japan with a stretched grid system in HRM and a uniform grid system in LRM for the present (the 2000) and the future (the 2030, as proposed by the Representative Concentrations Pathway 4.5, RCP4.5). These calculations were performed by nudging meteorological fields obtained from an atmosphere-ocean coupled model and providing emission inventories used in the coupled model. After correcting for bias, we calculated the excess mortality due to long-term exposure to PM2.5 for the elderly. Results showed the LRM underestimated by approximately 30 % (of PM2.5 concentrations in the 2000 and 2030), approximately 60 % (excess mortality in the 2000) and approximately 90 % (excess mortality in 2030) compared to the HRM results. The estimation of excess mortality therefore performed better with high-resolution grid sizes. In addition, we also found that our nesting method could be a useful tool to obtain better estimation results.

Rapid differentiation of citrus Hop stunt viroid variants by real-time RT-PCR and high resolution melting analysis.

PubMed

Loconsole, Giuliana; Onelge, Nuket; Yokomi, Raymond K; Kubaa, Raied Abou; Savino, Vito; Saponari, Maria

2013-01-01

The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVd-infected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI.

PubMed

Bester, Rachelle; Jooste, Anna E C; Maree, Hans J; Burger, Johan T

2012-09-27

Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.

PubMed

Pecavar, Verena; Blaschitz, Marion; Hufnagl, Peter; Zeinzinger, Josef; Fiedler, Anita; Allerberger, Franz; Maass, Matthias; Indra, Alexander

2012-06-01

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Effectiveness of Stereoscopic Displays for Indirect-Vision Driving and Robot Teleoperation

DTIC Science & Technology

2010-08-01

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Research Laboratory ATTN: RDRL- HRM -AT Aberdeen Proving Ground, MD 21005-5425 8...course of varying negative and positive terrain features (i.e., holes in the ground, drop- offs, and hills; figure 4c ). Participants were instructed...ORGANIZATION COPIES ORGANIZATION 31 1 ARMY RSCH LABORATORY – HRED RDRL HRM A J MARTIN MYER CENTER BLDG 2700 RM 2D311 FORT MONMOUTH NJ 07703

Evaluating the potential of a novel dual heat-pulse sensor to measure volumetric water use in grapevines under a range of flow conditions

USDA-ARS?s Scientific Manuscript database

The aim of this study was to validate dual sap flow sensors that combine two heat pulse techniques to measure volumetric water use over the full range of sap flows found in grapevines. The heat ratio method (HRM), which works well at measuring low and reverse flows, was combined with the compensati...

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

PubMed

Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

2013-11-13

In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Heat stress mortality and desired adaptation responses of healthcare system in Poland

NASA Astrophysics Data System (ADS)

Błażejczyk, Anna; Błażejczyk, Krzysztof; Baranowski, Jarosław; Kuchcik, Magdalena

2018-03-01

Heat stress is one of the environmental factors influencing the health of individuals and the wider population. There is a large body of research to document significant increases in mortality and morbidity during heat waves all over the world. This paper presents key results of research dealing with heat-related mortality (HRM) in various cities in Poland which cover about 25% of the country's population. Daily mortality and weather data reports for the years 1991-2000 were used. The intensity of heat stress was assessed by the universal thermal climate index (UTCI). The research considers also the projections of future bioclimate to the end of twenty-first century. Brain storming discussions were applied to find necessary adaptation strategies of healthcare system (HCS) in Poland, to minimise negative effects of heat stress. In general, in days with strong and very strong heat stress, ones must expect increase in mortality (in relation to no thermal stress days) of 12 and 47%, respectively. Because of projected rise in global temperature and heat stress frequency, we must expect significant increase in HRM to the end of twenty-first century of even 165% in comparison to present days. The results of research show necessity of urgent implementation of adaptation strategies to heat in HCS.

Long-term Outcomes of Patients With Normal or Minor Motor Function Abnormalities Detected by High-resolution Esophageal Manometry.

PubMed

Ravi, Karthik; Friesen, Laurel; Issaka, Rachel; Kahrilas, Peter J; Pandolfino, John E

2015-08-01

High-resolution manometry (HRM) expands recognition of minor esophageal motor abnormalities, but the clinical significance of these is unclear. We aimed to determine the outcomes of minor esophageal motor abnormalities. We reviewed HRM tracings from patients who underwent esophageal manometry at Northwestern Memorial Hospital from July 2004 through October 2005 by using the Chicago classification (version 2.0). We identified 301 patients with normal findings or minor manometric abnormalities (weak peristalsis, hypertensive peristalsis, frequent failed peristalsis, or rapid contractions with normal latency). Ninety-eight patients participated in a phone survey in which they were asked questions from the impact dysphagia questionnaire (mean follow-up period, 6 years 5 months). Of 301 patients assessed, 166 had normal findings from HRM, 82 had weak peristalsis, 34 had hypertensive peristalsis, 17 had frequent failed peristalsis, and 2 had rapid contractions with normal latency. The primary indications for HRM of dysphagia (44%) and gastroesophageal reflux disease (63%) were unrelated to manometric findings. There were no endoscopic or videofluoroscopic differences between patients with minor manometric abnormalities. Of 98 patients with follow-up, findings from HRM were normal in 63, weak peristalsis was observed in 23, hypertensive peristalsis was observed in 10, and frequent failed peristalsis was observed in 2. No patients underwent surgical myotomy, pneumatic dilation, or botulinum toxin injection. Use of proton pump inhibitors and rates of fundoplication were similar, regardless of manometric findings. Sixteen patients (16%) had significant dysphagia at follow-up; hypertensive peristalsis was the most likely to be symptomatic. Patients with normal and minor esophageal motor abnormalities report minimal symptoms and have few medical interventions related to esophageal dysfunction during long-term follow-up. Therefore, identification of normal and minor motor function is likely a good prognostic indicator. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

HRM and its effect on employee, organizational and financial outcomes in health care organizations.

PubMed

Vermeeren, Brenda; Steijn, Bram; Tummers, Lars; Lankhaar, Marcel; Poerstamper, Robbert-Jan; van Beek, Sandra

2014-06-17

One of the main goals of Human Resource Management (HRM) is to increase the performance of organizations. However, few studies have explicitly addressed the multidimensional character of performance and linked HR practices to various outcome dimensions. This study therefore adds to the literature by relating HR practices to three outcome dimensions: financial, organizational and employee (HR) outcomes. Furthermore, we will analyze how HR practices influence these outcome dimensions, focusing on the mediating role of job satisfaction. This study uses a unique dataset, based on the 'ActiZ Benchmark in Healthcare', a benchmark study conducted in Dutch home care, nursing care and care homes. Data from autumn 2010 to autumn 2011 were analyzed. In total, 162 organizations participated during this period (approximately 35% of all Dutch care organizations). Employee data were collected using a questionnaire (61,061 individuals, response rate 42%). Clients were surveyed using the Client Quality Index for long-term care, via stratified sampling. Financial outcomes were collected using annual reports. SEM analyses were conducted to test the hypotheses. It was found that HR practices are - directly or indirectly - linked to all three outcomes. The use of HR practices is related to improved financial outcomes (measure: net margin), organizational outcomes (measure: client satisfaction) and HR outcomes (measure: sickness absence). The impact of HR practices on HR outcomes and organizational outcomes proved substantially larger than their impact on financial outcomes. Furthermore, with respect to HR and organizational outcomes, the hypotheses concerning the full mediating effect of job satisfaction are confirmed. This is in line with the view that employee attitudes are an important element in the 'black box' between HRM and performance. The results underscore the importance of HRM in the health care sector, especially for HR and organizational outcomes. Further analyses of HRM in the health care sector will prove to be a productive endeavor for both scholars and HR managers.

Effects of Cue Reliability on Target Detection and Visual Scanning

DTIC Science & Technology

2010-12-01

instruction on how to rate his workload experience using the NASA-TLX. The participant then completed one 3-min practice ride in each of the four...participant practiced completing the NASA-TLX to rate his workload experience. 3.4.2 Test Procedures After a 5-min rest break, the test period...RDRL HRM A J MARTIN MYER CENTER BLDG 2700 RM 2D311 FORT MONMOUTH NJ 07703-5601 1 ARMY RSCH LABORATORY – HRED RDRL HRM C A DAVISON

Adaptive Integration and Optimization of Automated and Neural Processing Systems - Establishing Neural and Behavioral Benchmarks of Optimized Performance

DTIC Science & Technology

2012-07-01

detection only condition followed either face detection only or dual task, thus ensuring that participants were practiced in face detection before...1 ARMY RSCH LABORATORY – HRED RDRL HRM C A DAVISON 320 MANSCEN LOOP STE 115 FORT LEONARD WOOD MO 65473 2 ARMY RSCH LABORATORY...HRED RDRL HRM DI T DAVIS J HANSBERGER BLDG 5400 RM C242 REDSTONE ARSENAL AL 35898-7290 1 ARMY RSCH LABORATORY – HRED RDRL HRS

Human Factors Assessment of the U.S. Naval Research Laboratory Limb Protection Program (QuadGard Phase 3 Pre-Pilot Production Design)

DTIC Science & Technology

2011-09-01

NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Research Laboratory ATTN: RDRL- HRM Aberdeen Proving...user’s hand must be inserted toward the elbow area of the QuadGard III sleeve. This issue was overcome by equipment familiarization and practice ...ADELPHI MD 20783-1197 NO. OF NO. OF COPIES ORGANIZATION COPIES ORGANIZATION 17 1 ARMY RSCH LABORATORY – HRED RDRL HRM DW E REDDEN

The use of real-time polymerase chain reaction with high resolution melting (real-time PCR-HRM) analysis for the detection and discrimination of nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

PubMed

Filipiak, Anna; Hasiów-Jaroszewska, Beata

2016-04-01

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Workload Analysis of the Crew of the Abrams V2 SEP: Phase I Baseline IMPRINT Model

DTIC Science & Technology

2009-09-01

PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Research Laboratory ATTN: RDRL- HRM -B Aberdeen Proving Ground, MD 21005-5425 8...running a stop sign or a deer darting across the road, the high workload demands of driving become more apparent. The frequency of automobile accidents...OF COPIES ORGANIZATION COPIES ORGANIZATION 29 1 ARMY RSCH LABORATORY – HRED RDRL HRM A J MARTIN MYER CENTER BLDG 2700 RM 2D311

Identification of Trypanosoma cruzi discrete typing units (DTUs) through the implementation of a high-resolution melting (HRM) genotyping assay.

PubMed

Higuera, Sonia L; Guhl, Felipe; Ramírez, Juan David

2013-04-20

Chagas disease, caused by Trypanosoma cruzi, is a geographically widespread anthropozoonosis that is considered a major public health problem in Latin America. Because this parasite presents high genetic variability, a nomenclature has been adopted to classify the parasite into six discrete typing units (DTUs): TcI, TcII, TcIII, TcIV, TcV, and TcVI, which present different eco-epidemiological, clinical, and geographic associations. Currently, the available genotyping methods present a series of drawbacks that implies the need for developing new methods for characterizing T. cruzi DTU's. The aim of this work was to genotype reference populations from T. cruzi by means of a High-Resolution Melting (HRM) genotyping assay. Amplification of the mini-exon gene allowed the genotyping of three distinct groups: TcI, TcII- TcIV- TcV, and TcIII-TcVI, while amplification of the 24sα gene generated non-overlapping melting temperature ranges for each DTU that were used to identify the groups in the six existing DTUs of Trypanosoma cruzi. The proposed genotyping assay allowed discrimination of the six genetic groups by obtaining specific melting curves for each DTU. The application of this technique is proposed because of its specificity, sensitivity, high performance, and low cost compared with other previously described characterization methods.

Delta-Isobar Production in the Hard Photodisintegration of a Deuteron

NASA Astrophysics Data System (ADS)

Granados, Carlos; Sargsian, Misak

2010-02-01

Hard photodisintegration of the deuteron in delta-isobar production channels is proposed as a useful process in identifying the quark structure of hadrons and of hadronic interactions at large momentum and energy transfer. The reactions are modeled using the hard re scattering model, HRM, following previous works on hard breakup of a nucleon nucleon (NN) system in light nuclei. Here,quantitative predictions through the HRM require the numerical input of fits of experimental NN hard elastic scattering cross sections. Because of the lack of data in hard NN scattering into δ-isobar channels, the cross section of the corresponding photodisintegration processes cannot be predicted in the same way. Instead, the corresponding NN scattering process is modeled through the quark interchange mechanism, QIM, leaving an unknown normalization parameter. The observables of interest are ratios of differential cross sections of δ-isobar production channels to NN breakup in deuteron photodisintegration. Both entries in these ratios are derived through the HRM and QIM so that normalization parameters cancel out and numerical predictions can be obtained. )

Hard Break-Up of Two-Nucleons and QCD Dynamics of NN Interaction

NASA Astrophysics Data System (ADS)

Sargsian, Misak

2008-10-01

We discus recent developments in theory of high energy two-body break-up of few-nucleon systems. The characteristics of these reactions are such that the hard two-body quasielastic subprocess can be clearly separated from the accompanying soft subprocesses. We discuss in details the hard rescattering model (HRM) in which hard photodisintegration develops in two stages. At first, photon knocks-out an energetic quark which rescatters subsequently with a quark of the other nucleon. The latter provides a mechanism of sharing the initial high momentum of the photon between two outgoing nucleons. This final state hard rescattering can be expressed through the hard NN scattering amplitude. Within HRM we discuss hard break-up reactions involving D and 3He targets and demonstrate how these reactions are sensitive to the dynamics of hard pn and pp interaction. Another development of HRM is the prediction of new helicity selection mechanism for hard two-body reactions, which was apparently confirmed in the recent JLab experiment.

Bridge Over an Aging Population: Examining Longitudinal Relations Among Human Resource Management, Social Support, and Employee Outcomes Among Bridge Workers.

PubMed

Veth, Klaske N; Van der Heijden, Beatrice I J M; Korzilius, Hubert P L M; De Lange, Annet H; Emans, Ben J M

2018-01-01

This two-wave complete panel study aims to examine human resource management (HRM) bundles of practices in relation to social support [i.e., leader-member exchange (LMX), coworker exchange (CWX)] and employee outcomes (i.e., work engagement, employability, and health), within a context of workers aged 65+. Based upon the social exchange theory and the Job Demands-Resources (JD-R) framework, it was hypothesized that HRM bundles at Time 1 would increase bridge workers' outcomes at Time 2, and that this relationship would be mediated by perceptions of LMX and CWX at Time 2. Using a longitudinal design, hypotheses were tested in a unique sample of Dutch bridge employees ( N = 228). Results of several structural equation modeling analyses revealed no significant associations between HRM bundles, and social support, moreover, no significant associations were found in relation to employee outcomes. However, the results of the best-fitting final model revealed the importance of the impact of social support on employee (65+) outcomes over time.

Identification of TNIP1 Polymorphisms by High Resolution Melting Analysis with Unlabelled Probe: Association with Systemic Lupus Erythematosus

PubMed Central

Zhang, Jie; Chen, Yuewen; Shao, Yong; Wu, Qi; Guan, Ming; Zhang, Wei; Wan, Jun; Yu, Bo

2012-01-01

Background. TNFα-induced protein 3 (TNFAIP3) interacting with protein 1 (TNIP1) acts as a negative regulator of NF-κB and plays an important role in maintaining the homeostasis of immune system. A recent genome-wide association study (GWAS) showed that the polymorphism of TNIP1 was associated with the disease risk of SLE in Caucasian. In this study, we investigated whether the association of TNIP1 with SLE was replicated in Chinese population. Methods. The association of TNIP1 SNP rs7708392 (G/C) was determined by high resolution melting (HRM) analysis with unlabeled probe in 285 SLE patients and 336 healthy controls. Results. A new SNP rs79937737 located on 5 bp upstream of rs7708392 was discovered during the HRM analysis. No association of rs7708392 or rs79937737 with the disease risk of SLE was found. Furthermore, rs7708392 and rs79937737 were in weak linkage disequilibrium (LD). Hypotypes analysis of the two SNPs also showed no association with SLE in Chinese population. Conclusions. High resolution melting analysis with unlabeled probes proves to be a powerful and efficient genotyping method for identifying and screening SNPs. No association of rs7708392 or rs79937737 with the disease risk of SLE was observed in Chinese population. PMID:22852072

Thermodynamics of Meissner effect and flux pinning behavior in the bulk of single-crystal La 2 - x Sr x CuO 4 ( x = 0.09 )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhiman, I.; Ziesche, R.; Anand, V. K.

We have studied the magnetic flux pinning behavior and Meissner effect for the high-more » $$T_{\\rm c}$$ single crystal La$$_{2-x}$$Sr$$_{x}$$CuO$$_{4}$$ ($x$ = 0.09) superconductor using the polarized neutron imaging method with varying magnetic field and temperature. In the Meissner state expulsion of magnetic field (switched on during the measurements) is visualized, and the signatures of mixed state with increasing temperature are observed. While, for flux pinning behavior between 5 K $$\\leq$$ $T$ $$\\leq$$ 15 K and $$H_{\\rm ext}$$ = 63.5 mT (switched off during the measurements), the evolution of fringe pattern for both 0$$^{o}$$ and 90$$^{o}$$ sample orientation indicates magnetic flux pinning inside the bulk of the sample. At 25 K $$\\leq$$ $T$ $$\\leq$$ 32 K, a continuous decrease of inhomogeneously distribution pinned magnetic flux is observed, with the sample reaching a normal conducting state at $$T_{\\rm c}$$ ($$\\approx$$ 32 K). The flux pinning behavior is also explored as a function of $$H_{\\rm ext}$$, at $T$ = 5 K. As expected, with increasing $$H_{\\rm ext}$$ an increase in fringe density is observed, indicating an increase in magnetic flux pinning in the bulk of the sample. Therefore, in the present work for the first time we report bulk visualization of Meissner effect and flux pinning behavior in high-$$T_{\\rm c}$$ La$$_{2-x}$$Sr$$_{x}$$CuO$$_{4}$$ ($x$ = 0.09) superconductor. This study clearly demonstrates the potential of real space polarized neutron imaging technique for the visualization of the superconducting mixed state, particularly in the field of high-$$T_{\\rm c}$$ superconductors.« less

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research.

PubMed

Go, Young-Mi; Walker, Douglas I; Liang, Yongliang; Uppal, Karan; Soltow, Quinlyn A; Tran, ViLinh; Strobel, Frederick; Quyyumi, Arshed A; Ziegler, Thomas R; Pennell, Kurt D; Miller, Gary W; Jones, Dean P

2015-12-01

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. A major challenge for exposome research lies in the development of robust and affordable analytic procedures to measure the broad range of exposures and associated biologic impacts occurring over a lifetime. Biomonitoring is an established approach to evaluate internal body burden of environmental exposures, but use of biomonitoring for exposome research is often limited by the high costs associated with quantification of individual chemicals. High-resolution metabolomics (HRM) uses ultra-high resolution mass spectrometry with minimal sample preparation to support high-throughput relative quantification of thousands of environmental, dietary, and microbial chemicals. HRM also measures metabolites in most endogenous metabolic pathways, thereby providing simultaneous measurement of biologic responses to environmental exposures. The present research examined quantification strategies to enhance the usefulness of HRM data for cumulative exposome research. The results provide a simple reference standardization protocol in which individual chemical concentrations in unknown samples are estimated by comparison to a concurrently analyzed, pooled reference sample with known chemical concentrations. The approach was tested using blinded analyses of amino acids in human samples and was found to be comparable to independent laboratory results based on surrogate standardization or internal standardization. Quantification was reproducible over a 13-month period and extrapolated to thousands of chemicals. The results show that reference standardization protocol provides an effective strategy that will enhance data collection for cumulative exposome research. In principle, the approach can be extended to other types of mass spectrometry and other analytical methods. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research

PubMed Central

Go, Young-Mi; Walker, Douglas I.; Liang, Yongliang; Uppal, Karan; Soltow, Quinlyn A.; Tran, ViLinh; Strobel, Frederick; Quyyumi, Arshed A.; Ziegler, Thomas R.; Pennell, Kurt D.; Miller, Gary W.; Jones, Dean P.

2015-01-01

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. A major challenge for exposome research lies in the development of robust and affordable analytic procedures to measure the broad range of exposures and associated biologic impacts occurring over a lifetime. Biomonitoring is an established approach to evaluate internal body burden of environmental exposures, but use of biomonitoring for exposome research is often limited by the high costs associated with quantification of individual chemicals. High-resolution metabolomics (HRM) uses ultra-high resolution mass spectrometry with minimal sample preparation to support high-throughput relative quantification of thousands of environmental, dietary, and microbial chemicals. HRM also measures metabolites in most endogenous metabolic pathways, thereby providing simultaneous measurement of biologic responses to environmental exposures. The present research examined quantification strategies to enhance the usefulness of HRM data for cumulative exposome research. The results provide a simple reference standardization protocol in which individual chemical concentrations in unknown samples are estimated by comparison to a concurrently analyzed, pooled reference sample with known chemical concentrations. The approach was tested using blinded analyses of amino acids in human samples and was found to be comparable to independent laboratory results based on surrogate standardization or internal standardization. Quantification was reproducible over a 13-month period and extrapolated to thousands of chemicals. The results show that reference standardization protocol provides an effective strategy that will enhance data collection for cumulative exposome research. In principle, the approach can be extended to other types of mass spectrometry and other analytical methods. PMID:26358001

High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

PubMed Central

2012-01-01

Background Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. PMID:22429680

Human resources management for a hospital pharmacy department.

PubMed

Chase, P A

1989-06-01

The concepts of human resources management (HRM) are presented, and the application of HRM concepts to a hospital pharmacy department is described. Low salaries and poor working conditions had precipitated a mass exodus of pharmacists from a 650-bed, tertiary-care medical center. The newly hired director of pharmacy sought to rebuild the department by developing a three-stage HRM model consisting of needs forecasting, performance management, and advanced management systems. In the needs-forecasting stage, the strengths and weaknesses of departmental programs were determined through analysis of existing standards of practice, situational analysis, and financial analyses; the strengths and weaknesses of departmental employees were determined through the use of talent inventories, turnover analysis, analysis of time and leave records, reevaluation of the department's job classifications, performance and productivity evaluations, and productivity evaluations, and development of a philosophy of practice and mission statement. Needs and problems were addressed by examining each existing program and developing new policies and procedures, performance standards, quality assurance mechanisms, and productivity expectations. Personnel needs and problems were addressed by designing a system of differentiated career ladders, contracting with pharmacists for career moves, developing the skills of currently employed pharmacists, and implementing a succession planning model. The model has been in place for approximately three years and is beginning to yield the desired results. Application of HRM concepts to a hospital pharmacy department appears to have been successful in improving employee morale and in helping the department to meet goals of expanded and improved services.

[Differences between Chicago and traditional classifications in the diagnosis of esophageal motor disorders with high-resolution manometry and topography of esophageal pressure].

PubMed

Abreu-Y Abreu, A T; González Sánchez, C B; Villanueva Sáenz, E; Valdovinos Díaz, M A

2010-01-01

With the introduction of high resolution manometry (HRM) and esophageal topography a novel classification (Chicago Classification) has been proposed for the diagnosis of esophageal motor disorders (EMD). Clinical differences with the traditional classification are currently under evaluation. To investigate differences between the Chicago (CC) and traditional (TC) classifications in the diagnosis of EMD. Consecutive patients with indication for esophageal manometry were studied. HRM was performed with a 36 sensors solid-state catheter and Manoview software (V2.0).Conventional manometric tracings were analyzed by an investigator blinded to the results of HRM. Diagnosis by CC and CT were compared. Two hundred patients were studied, 106 (53%) of them women (53%) with a mean patient age of 43.4 (range 16 - 84) years. Preoperative evaluation for GERD 152 (76%) was the most frequent indication. Achalasia (8), scleroderma (2) and peristaltic dysfunction (60 vs. 59) were similarly diagnosed by CC and CT. Spastic disorders were more frequently identified by CC: nutcracker esophagus (NC) in 3, spastic NC in3 and segmental NC in 11 patients versus TC: NC 5. Three patients had spasm with CC and 1 with TC. Non specific motor disorder was diagnosed by TC and 2 patients had functional obstruction with CC. Hypotensive lower esophageal sphincter was identified in 63 patients with CC vs.57 with TC. Spastic disorders and functional obstruction were the EMD better identified by HRM and CC.

Expiratory muscle strength training evaluated with simultaneous high-resolution manometry and electromyography.

PubMed

Hutcheson, Katherine A; Hammer, Michael J; Rosen, Sarah P; Jones, Corinne A; McCulloch, Timothy M

2017-04-01

To examine feasibility of a simultaneous high-resolution pharyngeal manometry (HRM) and electromyography (EMG) experimental paradigm to detect swallowing-related patterns of palatal, laryngeal, and pharyngeal muscle activity during expiratory training. Technical report. Simultaneous HRM, surface submental, and intramuscular EMG were acquired in two healthy participants during five tasks: 10-cc water swallow, maximum expiratory pressure (MEP) testing, and expiratory muscle strength training (EMST) at three pressure levels (sham, 50%, and 75% MEP). Experimental conditions were feasible. Velopharyngeal closing pressure, palate EMG activity, and pharyngeal EMG activity increased as expiratory load increased. In contrast, thyroarytenoid EMG activity was low during the expiratory task, consistent with glottic opening during exhalation. Submental EMG patterns were more variable during expiratory tasks. Intraluminal air pressures recorded with HRM were correlated with measured expiratory pressures and target valve-opening pressures of the EMST device. Results suggest that a simultaneous HRM/EMG/EMST paradigm may be used to detect previously unquantified swallowing-related muscle activity during EMST, particularly in the palate and pharynx. Our approach and initial findings will be helpful to guide future hypothesis-driven studies and may enable investigators to evaluate other muscle groups active during these tasks. Defining mechanisms of action is a critical next step toward refining therapeutic algorithms using EMST and other targeted treatments for populations with dysphagia and airway disorders. 4. Laryngoscope, 127:797-804, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

High-intensity focused ultrasound (HIFU) treatment for uterine fibroids: a meta-analysis.

PubMed

Ji, Yongshuo; Hu, Kaimeng; Zhang, Yu; Gu, Lijun; Zhu, Junqiu; Zhu, Linglin; Zhu, Yanfei; Zhao, Hong

2017-12-01

High-intensity focused ultrasound (HIFU) is a non-invasive uterine-preserving treatment alternative to hysterectomy for women with fibroids. We performed this meta-analysis to evaluate the efficacy of HIFU in the treatment of women with symptomatic fibroids comparing it to other approaches including medical treatment with mifepristone (Mife), traditional surgery with myomectomy or hysterectomy (MYC/HRM), and radiofrequency ablation (RF). 16 studies with 1725 women were included. The pooled data of HIFU comparing it to other methods in terms of complete or partial response rate (CR/PR) was not significantly better, but in subgroup analysis, the response rate was significantly higher than Mife, significantly lower than RF and comparable to MYC/HRM, respectively. For the endpoints of safety, the superiority of HIFU compared to MYC/HMR or Mife was found to be significant in terms of pain/discomfort, fever, transfusion, genital tract, gastrointestinal tract, and anesthesia-related complications, while no superiority was identified for skin burn, urinary tract, and nervous system complications. These results suggest that HIFU treatment of uterine leiomyomas leads to clinical improvement with few significant clinical complications and adverse events.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

PubMed

Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

2016-01-01

Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively). Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.

Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes.

PubMed

Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Gratz, Jean; Banu, Sayera; Rahman, S M Mazidur; Ferdous, Sara Sabrina; Foongladda, Suporn; Boonlert, Duangjai; Ogarkov, Oleg; Zhdanova, Svetlana; Kibiki, Gibson; Heysell, Scott; Houpt, Eric

2015-02-24

Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics. Copyright © 2015 Pholwat et al.

Esophageal Motor Abnormalities in Patients With Scleroderma: Heterogeneity, Risk Factors, and Effects on Quality of Life.

PubMed

Crowell, Michael D; Umar, Sarah B; Griffing, W Leroy; DiBaise, John K; Lacy, Brian E; Vela, Marcelo F

2017-02-01

Systemic scleroderma (SSc) is associated with esophageal aperistalsis and hypotensive esophagogastric junction pressure, although there could be a gradation in esophageal motor dysfunction. We characterized esophageal motor function by high-resolution esophageal manometry (HRM) and assessed associations between SSc severity, health-related quality of life (HRQOL), and HRM findings in patients. We performed a prospective study of 200 patients with SSc and 102 patients without SSc (controls) who underwent HRM at Mayo Clinic Arizona from May 2006 through January 2015. We used data on integrated relaxation pressure, distal contractile integral, and distal latency to classify esophageal motility disorders according to the Chicago Classification v 3.0. A subset of subjects (n = 122) completed SSc-specific gastrointestinal symptom and HRQOL questionnaires. HRM findings, symptoms, and HRQOL data were compared among diffuse SSc, limited SSc, and control subjects. Categorical variables were compared by using the χ 2 or Fisher exact test; continuous variables were compared by using Mann-Whitney or Kruskal-Wallis test. Multivariable logistic regression was used to assess the association between severity of esophageal dysmotility and baseline clinical factors. Among patients with SSc, 83 had diffuse SSc (42%), and 117 had limited SSc (58%). Absent contractility was more frequent in patients with SSc than in controls (56% vs 13%; P < .001). HRM findings varied among the patients; absent contractility (56%) was the most frequent diagnosis, followed by normal motility (26%) and ineffective esophageal motility (10%). Classic scleroderma esophagus (esophagogastric junction pressure with absent contractility) was only observed in 33% of patients (34% with diffuse SSc vs 32% limited SSc) (P = .880). Severe esophageal dysmotility was associated with disease duration, interstitial lung disease, and higher gastrointestinal symptom scores (P < .001). HRQOL was decreased in patients with SSc and severe esophageal dysmotility. Although severe dysmotility is more common in patients with SSc than in controls, we observed the so-called scleroderma esophagus in only one-third of patients with SSc. Esophageal motor function appears to be heterogeneous in SSc. Esophageal dysmotility reduces HRQOL in patients with SSc. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Intrabolus pressure on high-resolution manometry distinguishes fibrostenotic and inflammatory phenotypes of eosinophilic esophagitis.

PubMed

Colizzo, J M; Clayton, S B; Richter, J E

2016-08-01

The aim of this investigation was to determine the motility patterns of inflammatory and fibrostenotic phenotypes of eosinophilic esophagitis (EoE) utilizing high-resolution manometry (HRM). Twenty-nine patients with a confirmed diagnosis of EoE according to clinicopathological criteria currently being managed at the Joy McCann Culverhouse Swallowing Center at the University of South Florida were included in the retrospective analysis. Only patients who completed HRM studies were included in the analysis. Patients were classified into inflammatory or fibrostenotic subtypes based on baseline endoscopic evidence. Their baseline HRM studies prior to therapy were analyzed. Manometric data including distal contractile integral, integrated relaxation pressure, and intrabolus pressure (IBP) values were recorded. HRM results were interpreted according to the Chicago Classification system. Statistical analysis was performed with SPSS software (Version 22, IBM Co., Armonk, NY, USA). Data were compared utilizing Student's t-test, χ(2) test, Pearson correlation, and Spearman correlation tests. Statistical significance was set at P < 0.05. A total of 29 patients with EoE were included into the retrospective analysis. The overall average age among patients was 40 years. Male patients comprised 62% of the overall population. Both groups were similar in age, gender, and overall clinical presentation. Seventeen patients (58%) had fibrostenotic disease, and 12 (42%) displayed inflammatory disease. The average IBP for the fibrostenotic and inflammatory groups were 18.6 ± 6.0 mmHg and 12.6 ± 3.5 mmHg, respectively (P < 0.05). Strictures were only seen in the fibrostenotic group. Of the fibrostenotic group, 6 (35%) demonstrated proximal esophageal strictures, 7 (41%) had distal strictures, 3 (18%) had mid-esophageal strictures, and 1 (6%) patient had pan-esophageal strictures. There was no statistically significant correlation between the level of esophageal stricture and degree of IBP. Integrated relaxation pressure, distal contractile integral, and other HRM metrics did not demonstrate statistical significance between the two subtypes. There also appeared no statistically significant correlation between patient demographics and esophageal metrics. Patients with the fibrostenotic phenotype of EoE demonstrated an IBP that was significantly higher than that of the inflammatory group. © 2015 International Society for Diseases of the Esophagus.

Disability management: organizational diversity and Dutch employment policy.

PubMed

Kopnina, Helen; Haafkens, Joke A

2010-06-01

While Human Resource Managers (HRM) and line managers could play a significant role in the prevention of job-related problems and in promotion of early job-continuation, it is not clear wether the chronically ill workers are recognized as a group. Unlike some other groups, distinguished by gender, age or ethnicity, those with chronic illness are less distinct and may not be included in diversity management programs. The aim of this research is to address theory and evidence in literature about the topic, as well as to inquire whether chronic illness of the employees is 'visible' in practice. For desk research, we used a systematic search strategy involving medical, statistical, management, and social science databases (Web of Science, MedLine, Pub Med, Psych Info, etc.). Research results are based on case studies conducted with the managers and HRM of government and commercial organizations between March 2007 and October 2008 and between October 2008 and April 2009. These case studies were based on open interviews and focus group sessions (for human resource departments) which were consequently analyzed using thematical analysis. For group sessions, we used concept mapping to collect information from two groups of HRM professionals and managers. Secondary analysis included thematic and content analysis of 'best practice' organizations carried out by the Dutch organization Gatekeeper. We have discovered that the chronically ill employees are largely invisible to HRM practitioners, line managers who do not always have the right instruments for implementation of the European or national frameworks. Most practitioners are unaware of the impact of chronic illness in their organizations and in employees work life.

High-Resolution Melting Curve Analysis of the 16S Ribosomal Gene to Detect and Identify Pathogenic and Saprophytic Leptospira species in Colombian Isolates

PubMed Central

Peláez Sánchez, Ronald G.; Quintero, Juan Álvaro López; Pereira, Martha María; Agudelo-Flórez, Piedad

2017-01-01

It is important to identify the circulating Leptospira agent to enhance the performance of serodiagnostic tests by incorporating specific antigens of native species, develop vaccines that take into account the species/serovars circulating in different regions, and optimize prevention and control strategies. The objectives of this study were to develop a polymerase chain reaction (PCR)–high-resolution melting (HRM) assay for differentiating between species of the genus Leptospira and to verify its usefulness in identifying unknown samples to species level. A set of primers from the initial region of the 16S ribosomal gene was designed to detect and differentiate the 22 species of Leptospira. Eleven reference strains were used as controls to establish the reference species and differential melting curves. Twenty-five Colombian Leptospira isolates were studied to evaluate the usefulness of the PCR–HRM assay in identifying unknown samples to species level. This identification was confirmed by sequencing and phylogenetic analysis of the 16S ribosomal gene. Eleven Leptospira species were successfully identified, except for Leptospira meyeri/Leptospira yanagawae because the sequences were 100% identical. The 25 isolates from humans, animals, and environmental water sources were identified as Leptospira santarosai (twelve), Leptospira interrogans (nine), and L. meyeri/L. yanagawae (four). The species verification was 100% concordant between PCR–HRM and phylogenetic analysis of the 16S ribosomal gene. The PCR–HRM assay designed in this study is a useful tool for identifying Leptospira species from isolates. PMID:28500802

Therapeutic Outcome of Achalasia Based on High-Resolution Manometry: A Korean Multicenter Study.

PubMed

Lee, Hyuk; Chung, Hyunsoo; Lee, Tae Hee; Hong, Kyoung Sup; Youn, Young Hoon; Park, Jung Ho; Park, Hyung Seok; Park, Hyojin

2017-09-11

Because achalasia subtype is associated with therapeutic response, it is possible that regional differences in subtype distribution could lead to differences in therapeutic outcomes. We aimed to evaluate and compare high-resolution manometry (HRM) profiles among the different subtypes of achalasia and to elucidate predictive factors associated with treatment outcomes. Patients who were diagnosed with achalasia using HRM at 4 Korean university hospitals were retrospectively identified and analyzed. Sixty-four patients with untreated achalasia were divided into 3 subtypes using the Chicago classification system. Clinical characteristics, manometric features, and treatment outcomes were compared. Among 64 patients diagnosed with achalasia, 31 patients were classified as type I, 27 as type II, and 6 as type III. Regarding HRM parameters, there were statistically significant differences in basal lower esophageal sphincter pressure, 4-second-integrated relaxation pressure, residual upper esophageal sphincter pressure, body amplitude, and maximal intrabolus pressure between subtypes. Regarding therapeutic outcome, type II patients (overall success rate of 80.0%) were more likely to respond than type I (55.2%) or type III (33.2%) patients. Multivariate analysis demonstrated that achalasia subtype (type I vs. III, P = 0.072; type II vs. III, P = 0.005), therapeutic modality (dilation vs. pharmacologic, P = 0.013; laparoscopic Heller's myotomy vs. pharmacologic, P = 0.006), and HRM-measured esophageal length (<27.5 vs. ≥27.5 cm, P = 0.014) are independent predictive factors for therapeutic failure. Patients with type II achalasia had better treatment outcomes than patients with other achalasia subtypes. Achalasia subtype, therapeutic modality, and esophageal length are independent predictive factors of therapeutic outcome.

Failed Deglutitive Upper Esophageal Sphincter Relaxation Is a Risk Factor for Aspiration in Stroke Patients with Oropharyngeal Dysphagia.

PubMed

Lee, Taeheon; Park, Jung Ho; Sohn, Chongil; Yoon, Kyung Jae; Lee, Yong-Taek; Park, Jung Hwan; Jung, Il Seok

2017-01-30

We attempted to examine the relationship between abnormal findings on high-resolution manometry (HRM) and videofluoroscopic swallowing study (VFSS) of the pharynx and upper esophageal sphincter (UES), and to identify the risk factors for aspiration. We performed VFSS and HRM on the same day in 36 ischemic stroke patients (mean age, 67.5 years) with dysphagia. Pressure (basal, median intra bolus, and nadir), relaxation time interval of the UES, and mesopharyngeal and hypopharyngeal contractility (as a contractile integral) were examined using HRM. The parameters of VFSS were vallecular residue, pyriform sinus residue, vallecular overflow, penetration, and aspiration. The association between the parameters of VFSS and HRM was analyzed by the Student's t test. Three (8.3%) and 4 (11.1%) stroke patients with dysphagia had pyriform sinus residue and vallecular sinus residue, respectively, and 5 (13.8%) patients showed aspiration. Mesopharyngeal and hypopharyngeal contractile integrals in patients with residue in the pyriform sinus were significantly lower than those in patients without residue in the pyriform sinus ( P < 0.05). Relaxation time intervals in patients with aspiration were significantly shorter than those in patients without aspiration ( P < 0.05), and multivariate regression analysis revealed a shorter relaxation time interval as the main risk factor for aspiration (OR, 0.03; 95% CI, 0.01-0.65; P < 0.05). Manometric measurements of the pharynx and UES were well correlated with abnormal findings in the VFSS, and a shorter relaxation time interval of the UES during deglutition is an important parameter for the development of aspiration.

Novel mutation of GATA4 gene in Kurdish population of Iran with nonsyndromic congenital heart septals defects.

PubMed

Soheili, Fariborz; Jalili, Zahra; Rahbar, Mahtab; Khatooni, Zahed; Mashayekhi, Amir; Jafari, Hossein

2018-03-01

The mutations in GATA4 gene induce inherited atrial and ventricular septation defects, which is the most frequent forms of congenital heart defects (CHDs) constituting about half of all cases. We have performed High resolution melting (HRM) mutation scanning of GATA4 coding exons of nonsyndrome 100 patients as a case group including 39 atrial septal defects (ASD), 57 ventricular septal defects (VSD) and four patients with both above defects and 50 healthy individuals as a control group. Our samples are categorized according to their HRM graph. The genome sequencing has been done for 15 control samples and 25 samples of patients whose HRM analysis were similar to healthy subjects for each exon. The PolyPhen-2 and MUpro have been used to determine the causative possibility and structural stability prediction of GATA4 sequence variation. The HRM curve analysis exhibit that 21 patients and 3 normal samples have deviated curves for GATA4 coding exons. Sequencing analysis has revealed 12 nonsynonymous mutations while all of them resulted in stability structure of protein 10 of them are pathogenic and 2 of them are benign. Also we found two nucleotide deletions which one of them was novel and one new indel mutation resulting in frame shift mutation, and 4 synonymous variations or polymorphism in 6 of patients and 3 of normal individuals. Six or about 50% of these nonsynonymous mutations have not been previously reported. Our results show that there is a spectrum of GATA4 mutations resulting in septal defects. © 2018 Wiley Periodicals, Inc.

Anxiety can significantly explain bolus perception in the context of hypotensive esophageal motility: Results of a large multicenter study in asymptomatic individuals.

PubMed

Cisternas, D; Scheerens, C; Omari, T; Monrroy, H; Hani, A; Leguizamo, A; Bilder, C; Ditaranto, A; Ruiz de León, A; Pérez de la Serna, J; Valdovinos, M A; Coello, R; Abrahao, L; Remes-Troche, J; Meixueiro, A; Zavala, M A; Marin, I; Serra, J

2017-09-01

Previous studies have not been able to correlate manometry findings with bolus perception. The aim of this study was to evaluate correlation of different variables, including traditional manometric variables (at diagnostic and extreme thresholds), esophageal shortening, bolus transit, automated impedance manometry (AIM) metrics and mood with bolus passage perception in a large cohort of asymptomatic individuals. High resolution manometry (HRM) was performed in healthy individuals from nine centers. Perception was evaluated using a 5-point Likert scale. Anxiety was evaluated using Hospitalized Anxiety and Depression scale (HAD). Subgroup analysis was also performed classifying studies into normal, hypotensive, vigorous, and obstructive patterns. One hundred fifteen studies were analyzed (69 using HRM and 46 using high resolution impedance manometry (HRIM); 3.5% swallows in 9.6% of volunteers were perceived. There was no correlation of any of the traditional HRM variables, esophageal shortening, AIM metrics nor bolus transit with perception scores. There was no HRM variable showing difference in perception when comparing normal vs extreme values (percentile 1 or 99). Anxiety but not depression was correlated with perception. Among hypotensive pattern, anxiety was a strong predictor of variance in perception (R 2 up to .70). Bolus perception is less common than abnormal motility among healthy individuals. Neither esophageal motor function nor bolus dynamics evaluated with several techniques seems to explain differences in bolus perception. Different mechanisms seem to be relevant in different manometric patterns. Anxiety is a significant predictor of bolus perception in the context of hypotensive motility. © 2017 John Wiley & Sons Ltd.

Diagnostic yield of 24-hour esophageal manometry in non-cardiac chest pain.

PubMed

Barret, M; Herregods, T V K; Oors, J M; Smout, A J P M; Bredenoord, A J

2016-08-01

In the past, ambulatory 24-h manometry has been shown useful for the evaluation of patients with non-cardiac chest pain (NCCP). With the diagnostic improvements brought by pH-impedance monitoring and high-resolution manometry (HRM), the contribution of ambulatory 24-h manometry to the diagnosis of esophageal hypertensive disorders has become uncertain. Our aim was to assess the additional diagnostic yield of ambulatory manometry to HRM and ambulatory pH-impedance monitoring in this patient population. All patients underwent 24-h ambulatory pressure-pH-impedance monitoring and HRM. Patients had retrosternal pain as a predominant symptom and no explanation after cardiologic and digestive endoscopic evaluations. Diagnostic measurements were analyzed by two independent physicians. Fifty-nine patients met the inclusion criteria; 37.3% of the patients had their symptoms explained by abnormalities on pH-impedance monitoring and 6.8% by ambulatory manometry. Functional chest pain was diagnosed in 52.5% of the patients. High-resolution manometry, using the Chicago Classification v3.0 criteria alone, did not identify any of the four patients with esophageal spasm on ambulatory manometry. However, taking into account other abnormalities, such as simultaneous (rapid) or repetitive contractions, HRM had a sensitivity of 75% and a specificity of 98.2% for the diagnosis of esophageal spasm. In the work-up of NCCP, ambulatory 24-h manometry has a low additional diagnostic yield. However, it remains the best technique to identify esophageal spasm as the cause of symptoms. This is particularly useful when an unequivocal diagnosis is needed before treatment. © 2016 John Wiley & Sons Ltd.

Determinants and clinical impact of pressure drift in manoscan anorectal high resolution manometry system.

PubMed

Parthasarathy, G; McMaster, J; Feuerhak, K; Zinsmeister, A R; Bharucha, A E

2016-09-01

Pressure drift (PD), resulting from differences between room and body temperature, reduces the accuracy of pressure measurements with the Manoscan high resolution manometry (HRM) system. Our aims were to assess PD during anorectal HRM. Defined as the residual pressure measured immediately after the catheter was removed, PD was calculated for each sensor and averaged across all 12 sensors in 454 anorectal consecutive studies recorded with 3 HRM catheters. The relationship between PD and study duration, number of prior uses of a catheter, and peak and average pressure exposure during a study were evaluated. The correction of PD with a software algorithm (thermal compensation) was evaluated in 76 studies where the most distal sensor was outside the body. The PD varied among sensors and across catheters. The average PD (7.3 ± 0.2 mmHg) was significantly greater for newer catheters, during longer studies, or when sensors were exposed to higher pressures. Together, these factors explained 81% of the variance in overall PD. After thermal compensation, the uncorrected median PD for the most distal sensor was 2.5-5 mmHg over the study duration. Correcting this changed the interpretation (e.g., as abnormal instead of normal) of at least 1 anorectal parameter in eight of 76 studies. During anorectal HRM, PD declines with catheter use and is greater for newer catheters, when sensors are exposed to higher pressures, and for studies of longer duration. While PD is partially corrected with thermal compensation algorithms, the impact on interpretation is modest. © 2016 John Wiley & Sons Ltd.

Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model.

PubMed

Hewson, Kylie; Noormohammadi, Amir H; Devlin, Joanne M; Mardani, Karim; Ignjatovic, Jagoda

2009-01-01

Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. > or =95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3'UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3'UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3'UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.

Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low DNA Content

PubMed Central

Milbury, Coren A.; Chen, Clark C.; Mamon, Harvey; Liu, Pingfang; Santagata, Sandro; Makrigiorgos, G. Mike

2011-01-01

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification. PMID:21354058

Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis.

PubMed

Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc; Maleewong, Wanchai

2013-12-01

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±0.09℃ and 85.9±0.08℃ 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.

Bridge Over an Aging Population: Examining Longitudinal Relations Among Human Resource Management, Social Support, and Employee Outcomes Among Bridge Workers

PubMed Central

Veth, Klaske N.; Van der Heijden, Beatrice I. J. M.; Korzilius, Hubert P. L. M.; De Lange, Annet H.; Emans, Ben J. M.

2018-01-01

This two-wave complete panel study aims to examine human resource management (HRM) bundles of practices in relation to social support [i.e., leader–member exchange (LMX), coworker exchange (CWX)] and employee outcomes (i.e., work engagement, employability, and health), within a context of workers aged 65+. Based upon the social exchange theory and the Job Demands-Resources (JD-R) framework, it was hypothesized that HRM bundles at Time 1 would increase bridge workers' outcomes at Time 2, and that this relationship would be mediated by perceptions of LMX and CWX at Time 2. Using a longitudinal design, hypotheses were tested in a unique sample of Dutch bridge employees (N = 228). Results of several structural equation modeling analyses revealed no significant associations between HRM bundles, and social support, moreover, no significant associations were found in relation to employee outcomes. However, the results of the best-fitting final model revealed the importance of the impact of social support on employee (65+) outcomes over time. PMID:29755386

An analytical model for regular respiratory signals derived from the probability density function of Rayleigh distribution.

PubMed

Li, Xin; Li, Ye

2015-01-01

Regular respiratory signals (RRSs) acquired with physiological sensing systems (e.g., the life-detection radar system) can be used to locate survivors trapped in debris in disaster rescue, or predict the breathing motion to allow beam delivery under free breathing conditions in external beam radiotherapy. Among the existing analytical models for RRSs, the harmonic-based random model (HRM) is shown to be the most accurate, which, however, is found to be subject to considerable error if the RRS has a slowly descending end-of-exhale (EOE) phase. The defect of the HRM motivates us to construct a more accurate analytical model for the RRS. In this paper, we derive a new analytical RRS model from the probability density function of Rayleigh distribution. We evaluate the derived RRS model by using it to fit a real-life RRS in the sense of least squares, and the evaluation result shows that, our presented model exhibits lower error and fits the slowly descending EOE phases of the real-life RRS better than the HRM.

ApoHRP-based assay to measure intracellular regulatory heme.

PubMed

Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A; Dhahbi, Joseph M

2015-02-01

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

Antioxidant and Nephroprotective Activities of the Extract and Fractions of Homonoia riparia Lour.

PubMed

Xavier, Seena Kanniparambil; Haneefa, Shoja Muhammed; Anand, Devkar Raviraj; Polo, Picheswara Rao; Maheshwari, Rajalekshmi; Shreedhara, Chandrashekara Shastry; Setty, Manganahalli Manjunath

2017-01-01

Homonoia riparia is a plant, which is widely used in the indigenous system of medicine for the treatment of urolithiasis, renal disorders and inflammatory conditions. This is the first report on the antioxidant and nephroprotective activities of whole plant of H. riparia . The present study aims at investigating the in vitro antioxidant and nephroprotective activity of the methanol extract and its different fractions of H. riparia . Petroleum ether (HRPE), Ethyl acetate (HREA), Butanol (HRBU), aqueous fractions (HRAQ) were prepared from the crude methanol extract of H. riparia (HRM) using liquid partitioning. Total phenolic content, flavonoid content and antioxidant activity assay were performed according to suitable methods. Nephroprotective activities were evaluated by MTT assay using Human Embryonic Kidney cells against cisplatin induced toxicity. Quantification of gallic acid was performed using validated HPTLC method. The studies showed that extract and fractions possess significant nephroprotective activity against cisplatin induced renal toxicity. All the extracts/fractions of whole plant of Homonoia riparia was found to be significantly reducing cisplatin induced toxicity (< 0.05). The highest activity was observed with HRBU and HRAQ with a percentage viability of 293.09 ± 4.3 and 345.07 ± 3.2 at a concentration of 200 µg/ml. Gallic acid was detected in the HRM/fractions using HPTLC. Cisplatin (8 μg/ml) exhibited 50 % inhibition in cell viability in HEK 293 cellsButanol and aqueous fractions of Homonoia riparia showed significant nephroprotective activity against cisplatin induced cell damage in HEK cells.Gallic acid was detected and quantified in the extract and fractions of whole plant of Homonoia riparia Abbreviations used: HPTLC: High Performance Thin Layer Chromatography, DPPH: 1,1-diphenyl-2-picrylhydrazyl, ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, MTT: 3-(4,5-dimethylthiazolyl-2-yl)-2,5- diphenyl tetrazolium bromide, GAE: Gallic acid equivalents, QE: Quercetin equivalents, HEK: Human Embryonic Kidney, HRM: Methanol extract of H. riparia, HRPE: Petroleum ether fraction of H. riparia, HREA: ethyl acetate fraction of H. riparia, HRBU: Butanol fraction of H. riparia, HRAQ: Aqueous fraction of H. riparia, DMEM: Dulbecco's minimum essential medium, FBS: Foetal bovine serum, DMSO: Dimethyl sulfoxide, ANOVA: One way analysis of variance.

High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

PubMed

Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

2016-02-01

Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

PubMed

Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

2012-01-01

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

A Rare Missense Mutation and a Polymorphism with High Frequency in LDLR Gene among Iranian Patients with Familial Hypercholesterolemia

PubMed Central

Tajamolian, Masoud; Kolahdouz, Parisa; Nikpour, Parvaneh; Forouzannia, Seyed Khalil; Sheikhha, Mohammad Hasan; Yazd, Ehsan Farashahi

2018-01-01

Background: Familial hypercholesterolemia (FH) is a disorder that is inherited by autosomal dominant pattern. The main cause of FH disease is the occurrence of mutations in low-density lipoprotein receptor (LDLR) gene sequence, as well as apolipoprotein B and proprotein convertase subtilisin/kexin type 9 genes, located in the next ranks, respectively. Materials and Methods: Forty-five unrelated Iranian patients with FH were screened using a high-resolution melting (HRM) method for exon 9 along with intron/exon boundaries of LDLR gene. Samples with shift in resultant HRM curves were compared to normal ones, sequenced, and analyzed. Results: Our findings revealed a missense mutation c. 1246C>T and a known variant IVS9-30C>T (rs1003723) that was recognized in 71% of the patients (22%: homozygous and 49%: heterozygous genotypes). In silico analysis, predicted the pathological effect of the c. 1246C>T mutation in LDLR protein structure, but IVS9-30C>T variant had no predicted effect on splice site and branch point function. Conclusion: FH is a hereditary type of hypercholesterolemia that leads to premature cardiovascular disease and atherosclerosis, and early diagnosis is needed. We detected a rare missense mutation (1246C>T) and a common single nucleotide polymorphism (SNP) in the Iranian population. These reports could help in the genetic diagnosis and counseling of FH patients. PMID:29531935

Evaluation and management of esophageal manifestations in systemic sclerosis.

PubMed

Denaxas, Konstantinos; Ladas, Spyros D; Karamanolis, George P

2018-01-01

Systemic sclerosis (SSc) is a multisystemic autoimmune connective tissue disorder; in the gastrointestinal tract, the esophagus is the most commonly affected organ. Symptoms of esophageal disease are due to gastroesophageal reflux disease (GERD) and esophageal motor dysfunction. Since the development of high-resolution manometry (HRM), this method has been preferred for the study of SSc patients with esophageal involvement. Using HRM, classic scleroderma esophagus, defined as absent or ineffective peristalsis of the distal esophagus in combination with a hypotensive lower esophageal sphincter, was found in as many as 55% of SSc patients. Endoscopy is the appropriate test for evaluating dysphagia and identifying evidence and possible complications of GERD. In the therapeutic area, treatment ranges from general supportive measures to the administration of drugs such as proton pump inhibitors and/or prokinetics. However, as many SSc patients do not respond to existing therapies, there is an urgent need for new therapeutic modalities. Buspirone, a 5-hydroxytryptamine 1A receptor agonist, could be a putative therapeutic option, as it was found to exert a significant beneficial effect in SSc patients with esophageal involvement. This review summarizes our knowledge concerning the evaluation and management of esophageal manifestations in SSc patients, including emerging therapeutic modalities.

Evaluation and management of esophageal manifestations in systemic sclerosis

PubMed Central

Denaxas, Konstantinos; Ladas, Spyros D.; Karamanolis, George P.

2018-01-01

Systemic sclerosis (SSc) is a multisystemic autoimmune connective tissue disorder; in the gastrointestinal tract, the esophagus is the most commonly affected organ. Symptoms of esophageal disease are due to gastroesophageal reflux disease (GERD) and esophageal motor dysfunction. Since the development of high-resolution manometry (HRM), this method has been preferred for the study of SSc patients with esophageal involvement. Using HRM, classic scleroderma esophagus, defined as absent or ineffective peristalsis of the distal esophagus in combination with a hypotensive lower esophageal sphincter, was found in as many as 55% of SSc patients. Endoscopy is the appropriate test for evaluating dysphagia and identifying evidence and possible complications of GERD. In the therapeutic area, treatment ranges from general supportive measures to the administration of drugs such as proton pump inhibitors and/or prokinetics. However, as many SSc patients do not respond to existing therapies, there is an urgent need for new therapeutic modalities. Buspirone, a 5-hydroxytryptamine 1A receptor agonist, could be a putative therapeutic option, as it was found to exert a significant beneficial effect in SSc patients with esophageal involvement. This review summarizes our knowledge concerning the evaluation and management of esophageal manifestations in SSc patients, including emerging therapeutic modalities. PMID:29507463

Larvicidal Activities of Indigenous Bacillus thuringiensis Isolates and Nematode Symbiotic Bacterial Toxins against the Mosquito Vector, Culex pipiens (Diptera: Culicidae).

PubMed

Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A

2017-06-01

The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens . B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC 50 and LC 95 ) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6-5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC 50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases.

High-resolution Manometry and Globus: Comparison of Globus, Gastroesophageal Reflux Disease and Normal Controls Using High-resolution Manometry.

PubMed

Choi, Won Seok; Kim, Tae Wan; Kim, Ja Hyun; Lee, Sang Hyuk; Hur, Woon Je; Choe, Young Gil; Lee, Sang Hyuk; Park, Jung Ho; Sohn, Chong Il

2013-10-01

Globus is a foreign body sense in the throat without dysphagia, odynophagia, esophageal motility disorders, or gastroesophageal reflux. The etiology is unclear. Previous studies suggested that increased upper esophageal sphincter pressure, gastroesophageal reflux and hypertonicity of esophageal body were possible etiologies. This study was to quantify the upper esophageal sphincter (UES) pressure, contractile front velocity (CFV), proximal contractile integral (PCI), distal contractile integral (DCI) and transition zone (TZ) in patient with globus gastroesophageal reflux disease (GERD) without globus, and normal controls to suggest the correlation of specific high-resolution manometry (HRM) findings and globus. Fifty-seven globus patients, 24 GERD patients and 7 normal controls were studied with HRM since 2009. We reviewed the reports, and selected 5 swallowing plots suitable for analysis in each report, analyzed each individual plot with ManoView. The 5 parameters from each plot in 57 globus patients were compared with that of 24 GERD patients and 7 normal controls. There was no significant difference in the UES pressure, CFV, PCI and DCI. TZ (using 30 mmHg isobaric contour) in globus showed significant difference compared with normal controls and GERD patients. The median values of TZ were 4.26 cm (interquartile range [IQR], 2.30-5.85) in globus patients, 5.91 cm (IQR, 3.97-7.62) in GERD patients and 2.26 cm (IQR, 1.22-2.92) in normal controls (P = 0.001). HRM analysis suggested that UES pressure, CFV, PCI and DCI were not associated with globus. Instead increased length of TZ may be correlated with globus. Further study comparing HRM results in globus patients within larger population needs to confirm their correlation.

Evaluating the link between human resource management decisions and patient satisfaction with quality of care.

PubMed

Oppel, Eva-Maria; Winter, Vera; Schreyögg, Jonas

Patient satisfaction with quality of care is becoming increasingly important in the competitive hospital market. Simultaneously, the growing shortage of clinical staff poses a considerable challenge to ensuring a high quality of care. In this context, a question emerges regarding whether and how human resource management (HRM) might serve as a means to reduce staff shortage problems and to increase patient satisfaction. Although considerable efforts have been devoted to understanding the concepts of patient satisfaction and HRM, little is known about the interrelationships between these concepts or about the link between staff shortage problems and patients' satisfaction with quality of care. The aim of this study was to investigate the relationship between strategic human resource management (SHRM), staff shortage problems, and patients' satisfaction with care. Furthermore, we analyze how the HRM decision to fill short-term vacancies through temporary staffing affects patient satisfaction. We differentiate between physicians and nurses. We develop and empirically test a theoretical model. The data (n = 165) are derived from a survey on SHRM that was sent to 732 German hospitals and from a survey on patient satisfaction that comprises 436,848 patient satisfaction ratings. We use a structural equation modeling approach to test the model. The results indicate that SHRM significantly reduces staff shortage problems for both occupational groups. Having fewer physician shortage problems is significantly associated with higher levels of patient satisfaction, whereas this effect is not significant for nurses. Furthermore, the use of temporary staffing considerably reduces patients' satisfaction with care. Hospital managers are advised to consider the effects of HRM decisions on patients' satisfaction with care. In particular, investments in SHRM targeted at physicians have significantly positive effects on patient satisfaction, whereas the temporary staffing of physicians and nurses should be avoided.

Esophageal contractions in type 3 achalasia esophagus: simultaneous or peristaltic?

PubMed Central

Kim, Tae Ho; Patel, Nirali; Ledgerwood-Lee, Melissa

2016-01-01

Absence of peristalsis and impaired relaxation of lower esophageal sphincter are the hallmarks of achalasia esophagus. Based on the pressurization patterns, achalasia has been subdivided into three subtypes. The goal of our study was to evaluate the esophageal contraction pattern and bolus clearance in type 3 achalasia esophagus. High-resolution manometry (HRM) recordings of all patients diagnosed with achalasia esophagus in our center between the years 2011 and 2013 were reviewed. Recordings of 36 patients with type 3 achalasia were analyzed for the characteristics of swallow-induced “simultaneous esophageal contraction.” The HRM impedance recordings of 14 additional patients with type 3 achalasia were analyzed for bolus clearance from the impedance recording. Finally, the HRM impedance along with intraluminal ultrasound imaging was conducted in six patients to further characterize the simultaneous esophageal contractions. Among 187 achalasia patients, 30 were type 1, 121 type 2, and 36 type 3. A total of 434 swallows evaluated in type 3 achalasia patients revealed that 95% of the swallow-induced contractions met criteria for simultaneous esophageal contraction, based on the onset of contraction. Interestingly, the peak and termination of the majority of simultaneous esophageal contractions were sequential. The HRM impedance revealed that 94% of the “simultaneous contractions” were associated with complete bolus clearance. Ultrasound image analysis revealed that baseline muscle thickness of patients in type 3 achalasia is larger than normal but the pattern of axial shortening is similar to that in normal subjects. The majority of esophageal contractions in type 3 achalasia are not true simultaneous contractions because the peak and termination of contraction are sequential and they are associated with complete bolus clearance. PMID:26950858

Esophageal contractions in type 3 achalasia esophagus: simultaneous or peristaltic?

PubMed

Kim, Tae Ho; Patel, Nirali; Ledgerwood-Lee, Melissa; Mittal, Ravinder K

2016-05-01

Absence of peristalsis and impaired relaxation of lower esophageal sphincter are the hallmarks of achalasia esophagus. Based on the pressurization patterns, achalasia has been subdivided into three subtypes. The goal of our study was to evaluate the esophageal contraction pattern and bolus clearance in type 3 achalasia esophagus. High-resolution manometry (HRM) recordings of all patients diagnosed with achalasia esophagus in our center between the years 2011 and 2013 were reviewed. Recordings of 36 patients with type 3 achalasia were analyzed for the characteristics of swallow-induced "simultaneous esophageal contraction." The HRM impedance recordings of 14 additional patients with type 3 achalasia were analyzed for bolus clearance from the impedance recording. Finally, the HRM impedance along with intraluminal ultrasound imaging was conducted in six patients to further characterize the simultaneous esophageal contractions. Among 187 achalasia patients, 30 were type 1, 121 type 2, and 36 type 3. A total of 434 swallows evaluated in type 3 achalasia patients revealed that 95% of the swallow-induced contractions met criteria for simultaneous esophageal contraction, based on the onset of contraction. Interestingly, the peak and termination of the majority of simultaneous esophageal contractions were sequential. The HRM impedance revealed that 94% of the "simultaneous contractions" were associated with complete bolus clearance. Ultrasound image analysis revealed that baseline muscle thickness of patients in type 3 achalasia is larger than normal but the pattern of axial shortening is similar to that in normal subjects. The majority of esophageal contractions in type 3 achalasia are not true simultaneous contractions because the peak and termination of contraction are sequential and they are associated with complete bolus clearance.

Esophageal peristaltic defects in adults with functional dysphagia.

PubMed

Ratuapli, Shiva K; Hansel, Stephanie L; Umar, Sarah B; Burdick, George E; Ramirez, Francisco C; Fleischer, David E; Harris, Lucinda A; Lacy, Brian E; DiBaise, John K; Crowell, Michael D

2014-08-01

Functional dysphagia (FD) is characterized by the presence of dysphagia without evidence of mechanical esophageal obstruction, GERD, and histopathology-based esophageal motor disorders. Dysphagia is common in older patients; however, there is a paucity of information regarding the type and frequency of peristaltic abnormalities compared to younger patients. Based on recently validated criteria for classification of weak peristalsis using high-resolution manometry (HRM), we hypothesized that older patients with FD would have more peristaltic defects detected by HRM compared to younger FD patients. A retrospective review of our motility database yielded 65 patients that met inclusion criteria. Patients were divided into two groups based on age (younger: <70 years; older: ≥70 years). Patients were interviewed, completed a quality-of-life questionnaire, and underwent solid-state HRM. The two groups differed in age but in no other demographic characteristics, severity of dysphagia, or quality of life. Dyspeptic symptoms, including nausea (p < 0.001), early satiety (p = 0.01), bloating (p = 0.02), and belching (p = 0.01), were also more prevalent in younger FD patients. Older age was associated with weak peristalsis involving frequent failed peristalsis, small proximal peristaltic defects (2-5 cm), and large proximal peristaltic defects (>5 cm) (p < 0.001). The mean contraction amplitude was also lower in the older group (p < 0.05). These data support the hypothesis that older patients with FD have a higher frequency of peristaltic abnormalities on HRM compared to younger patients. Older age was associated with increased frequency of weak peristalsis with small and large peristaltic defects.

Intra- and interrater reliability of the Chicago Classification of achalasia subtypes in pediatric high-resolution esophageal manometry (HRM) recordings.

PubMed

Singendonk, M M J; Rosen, R; Oors, J; Rommel, N; van Wijk, M P; Benninga, M A; Nurko, S; Omari, T I

2017-11-01

Subtyping achalasia by high-resolution manometry (HRM) is clinically relevant as response to therapy and prognosis have shown to vary accordingly. The aim of this study was to assess inter- and intrarater reliability of diagnosing achalasia and achalasia subtyping in children using the Chicago Classification (CC) V3.0. Six observers analyzed 40 pediatric HRM recordings (22 achalasia and 18 non-achalasia) twice by using dedicated analysis software (ManoView 3.0, Given Imaging, Los Angeles, CA, USA). Integrated relaxation pressure (IRP4s), distal contractile integral (DCI), intrabolus pressurization pattern (IBP), and distal latency (DL) were extracted and analyzed hierarchically. Cohen's κ (2 raters) and Fleiss' κ (>2 raters) and the intraclass correlation coefficient (ICC) were used for categorical and ordinal data, respectively. Based on the results of dedicated analysis software only, intra- and interrater reliability was excellent and moderate (κ=0.89 and κ=0.52, respectively) for differentiating achalasia from non-achalasia. For subtyping achalasia, reliability decreased to substantial and fair (κ=0.72 and κ=0.28, respectively). When observers were allowed to change the software-driven diagnosis according to their own interpretation of the manometric patterns, intra- and interrater reliability increased for diagnosing achalasia (κ=0.98 and κ=0.92, respectively) and for subtyping achalasia (κ=0.79 and κ=0.58, respectively). Intra- and interrater agreement for diagnosing achalasia when using HRM and the CC was very good to excellent when results of automated analysis software were interpreted by experienced observers. More variability was seen when relying solely on the software-driven diagnosis and for subtyping achalasia. Therefore, diagnosing and subtyping achalasia should be performed in pediatric motility centers with significant expertise. © 2017 John Wiley & Sons Ltd.

Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis.

PubMed

Kummalue, Tanawan; Chuphrom, Anchalee; Sukpanichanant, Sanya; Pongpruttipan, Tawatchai; Sukpanichanant, Sathien

2010-05-19

Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma. Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation 34. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis 56.GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost 7. Therefore, rapid and more cost effective technique are being sought.LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye 89. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product 1011. Therefore, current investigations using melting curve analysis are being developed 1213.In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.

The Cost Growth Problem: A Realistic Diagnosis and Solution.

DTIC Science & Technology

1983-06-01

access 30 0~ LJ 0 CD 0 ( 4c CD - V2 0 C.2 ~~CD.------ 0.0 4-J V) 0 0.0 C.,I Cdc = CC, 0 0D C2 =I . 31 haua La OC I- c C _g .- C2 n C2 __. 6.34 LIA ca D...Department Navy Military Personnel Command Washington, D.C. 20370 6. Director for HRM Plans and Policy (OP-IS0) 1 Human Resource Management Division...Washington, D.C. 20301 25. Dana French, CAPT, USN HD HRM Plans & Policy Branch Washington, D.C. 20301 26. Robert Aaron 820 Lamberton Drive Silver

A flexible, highly sensitive catheter for high resolution manometry based on in-fibre Bragg gratings

NASA Astrophysics Data System (ADS)

Bueley, Christopher; Wild, Peter M.

2013-09-01

This work presents a fibre optic-based flexible catheter for high resolution manometry (HRM), with sensing pods located at a pitch of 10 mm and an overall diameter of 2.8 mm. In-fibre Bragg gratings act as the sensing elements within these sensing pods. Hydrodynamic pressure resolution of 0.2 mmHg is demonstrated in conjunction with insensitivity to occlusion pressure. This result is significant in the context of HRM where independent measurement of hydrodynamic pressure is clinically relevant. The sensing system is compact, robust and flexible. Crosstalk between individual sensors is characterized and a compensation scheme is developed and validated.

Hard photodisintegration of 3He into a p d pair

NASA Astrophysics Data System (ADS)

Maheswari, Dhiraj; Sargsian, Misak M.

2017-02-01

The recent measurements of high energy photodisintegration of a 3He nucleus to a p d pair at 90∘ center of mass demonstrated an energy scaling consistent with the quark counting rule with an unprecedentedly large exponent of s-17. To understand the underlying mechanism of this process, we extended the theoretical formalism of the hard rescattering mechanism (HRM) to calculate the γ 3He→p d reaction. In HRM the incoming high energy photon strikes a quark from one of the nucleons in the target which subsequently undergoes hard rescattering with the quarks from the other nucleons, generating a hard two-body system in the final state of the reaction. Within the HRM we derived the parameter-free expression for the differential cross section of the reaction, which is expressed through the 3He→p d transition spectral function, the cross section of hard p d →p d scattering, and the effective charge of the quarks being interchanged during the hard rescattering process. The numerical estimates of all these factors resulted in the magnitude of the cross section, which is surprisingly in good agreement with the data.

Estimation of excess mortality due to long-term exposure to PM2.5 in Japan using a high-resolution model for present and future scenarios

NASA Astrophysics Data System (ADS)

Goto, Daisuke; Ueda, Kayo; Ng, Chris Fook Sheng; Takami, Akinori; Ariga, Toshinori; Matsuhashi, Keisuke; Nakajima, Teruyuki

2016-09-01

Particulate matter with a diameter of less than 2.5 μm, known as PM2.5, can affect human health, especially in elderly people. Because of the imminent aging of society in the near future in most developed countries, the human health impacts of PM2.5 must be evaluated. In this study, we used a global-to-regional atmospheric transport model to simulate PM2.5 in Japan with a high-resolution stretched grid system (∼10 km for the high-resolution model, HRM) for the present (the 2000) and the future (the 2030, as proposed by the Representative Concentrations Pathway 4.5, RCP4.5). We also used the same model with a low-resolution uniform grid system (∼100 km for the low-resolution model, LRM). These calculations were conducted by nudging meteorological fields obtained from an atmosphere-ocean coupled model and providing emission inventories used in the coupled model. After correcting for bias, we calculated the excess mortality due to long-term exposure to PM2.5 among the elderly (over 65 years old) based on different minimum PM2.5 concentration (MINPM) levels to account for uncertainty using the simulated PM2.5 distributions to express the health effect as a concentration-response function. As a result, we estimated the excess mortality for all of Japan to be 31,300 (95% confidence intervals: 20,700 to 42,600) people in 2000 and 28,600 (95% confidence intervals: 19,000 to 38,700) people in 2030 using the HRM with a MINPM of 5.8 μg/m3. In contrast, the LRM resulted in underestimates of approximately 30% (for PM2.5 concentrations in the 2000 and 2030), approximately 60% (excess mortality in the 2000) and approximately 90% (excess mortality in 2030) compared to the HRM results. We also found that the uncertainty in the MINPM value, especially for low PM2.5 concentrations in the future (2030) can cause large variability in the estimates, ranging from 0 (MINPM of 15 μg/m3 in both HRM and LRM) to 95,000 (MINPM of 0 μg/m3 in HRM) people.

Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

PubMed

Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

2015-01-01

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

Combustion performance and scale effect from N2O/HTPB hybrid rocket motor simulations

NASA Astrophysics Data System (ADS)

Shan, Fanli; Hou, Lingyun; Piao, Ying

2013-04-01

HRM code for the simulation of N2O/HTPB hybrid rocket motor operation and scale effect analysis has been developed. This code can be used to calculate motor thrust and distributions of physical properties inside the combustion chamber and nozzle during the operational phase by solving the unsteady Navier-Stokes equations using a corrected compressible difference scheme and a two-step, five species combustion model. A dynamic fuel surface regression technique and a two-step calculation method together with the gas-solid coupling are applied in the calculation of fuel regression and the determination of combustion chamber wall profile as fuel regresses. Both the calculated motor thrust from start-up to shut-down mode and the combustion chamber wall profile after motor operation are in good agreements with experimental data. The fuel regression rate equation and the relation between fuel regression rate and axial distance have been derived. Analysis of results suggests improvements in combustion performance to the current hybrid rocket motor design and explains scale effects in the variation of fuel regression rate with combustion chamber diameter.

Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

PubMed

Schoener, E R; Hunter, S; Howe, L

2017-07-01

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

PubMed

Boyd, Elaine M; Bench, Anthony J; Goday-Fernández, Andrea; Anand, Shubha; Vaghela, Krishna J; Beer, Phillip; Scott, Mike A; Bareford, David; Green, Anthony R; Huntly, Brian; Erber, Wendy N

2010-04-01

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Hard Photodisintegration of 3He

NASA Astrophysics Data System (ADS)

Granados, Carlos

2011-02-01

Large angle photodisintegration of two nucleons from the 3He nucleus is studied within the framework of the hard rescattering model (HRM). In the HRM the incoming photon is absorbed by one nucleon's valence quark that then undergoes a hard rescattering reaction with a valence quark from the second nucleon producing two nucleons emerging at large transverse momentum . Parameter free cross sections for pp and pn break up channels are calculated through the input of experimental cross sections on pp and pn elastic scattering. The calculated cross section for pp breakup and its predicted energy dependency are in good agreement with recent experimental data. Predictions on spectator momentum distributions and helicity transfer are also presented.

High-risk syndrome for neuromyelitis optica: a descriptive and comparative study.

PubMed

Collongues, N; Marignier, R; Zéphir, H; Blanc, F; Vukusic, S; Outteryck, O; Fleury, M; Ruet, A; Borgel, F; Thouvenot, E; Moreau, T; Defer, G; Derache, N; Pelletier, J; Audoin, B; Debouverie, M; Labauge, P; Gout, O; Camu, W; Brassat, D; Brochet, B; Vermersch, P; Confavreux, C; de Seze, J

2011-06-01

Neuromyelitis optica (NMO) frequently begins with a monofocal episode of optic neuritis or myelitis. A concept named high-risk syndrome (HRS) for NMO has been proposed for patients with monofocal episodes and NMO-IgG antibodies. To describe HRS patients and compare them with NMO patients. We identified 30 patients with HRS: 18 with extensive myelitis (HRM) and 12 with optic neuritis (HRON), in a database pooling patients from 25 centres in France. Clinical, laboratory/magnetic resonance imaging (MRI) data and outcome were analysed and compared with a national cohort of 125 NMO patients extracted from the same database. Mean follow-up was 4.8 years. Mean age at onset was 42.8 years (range: 12.4-70) with a female:male ratio of 0.9. Asymptomatic lesions were report on visual evoked potentials in 4/8 tested HRM patients and on spinal cord MRI in 2/7 HRON patients. Three patients died, two owing to a cervical lesion. HRS and NMO patients had similar clinical/paraclinical data, except for a predominance of men in the HRS group and a later mean age at onset in the HRM subgroup. The description of HRS patients is compatible with a monofocal form of NMO. Asymptomatic lesions could be included in a new set of NMO diagnostic criteria.

Backflow prevention mechanism of laparoscopic Toupet fundoplication using high-resolution manometry.

PubMed

Hoshino, Masato; Omura, Nobuo; Yano, Fumiaki; Tsuboi, Kazuto; Yamamoto, Se Ryung; Akimoto, Shunsuke; Kashiwagi, Hideyuki; Yanaga, Katsuhiko

2016-07-01

The use of multichannel intraluminal impedance pH (MII-pH) and high-resolution manometry (HRM), which are new devices used to examine the esophageal function, has recently become common in Europe and the USA, thus garnering much attention. There have not been enough studies as of yet, however, on the esophageal motor function and the benefits of treatment after these devices have been used in laparoscopic fundoplication. To use MII-pH and HRM to study the treatment effectiveness of laparoscopic fundoplication and consider a backflow prevention mechanism for laparoscopic Toupet fundoplication. The study looked at 27 of a total of 60 patients undergoing laparoscopic fundoplication due to reasons of either gastroesophageal reflux disease or esophageal hiatal hernia between October 2012 and February 2014, who underwent a postsurgical HRM examination. Of these, 25 patients whose symptoms disappeared following surgery and who were not orally administered gastric secretion inhibitor (of whom nine were male, average age 55.9 ± 14.9 years, and of whom 76 % underwent MII-pH) were taken as the subjects of the study. The postsurgical evaluation was conducted 3 months after the operation. Using HRM, although no change was noted in the lower esophageal sphincter pressure (LESP) (p = 0.943), an increase in lower esophageal sphincter pressure integral (p = 0.024) and extensions in both overall length and abdominal length were noted (both p < 0.001), while a significant improvement was noted in the lower esophageal sphincter (LES). Furthermore, the cases subjected to MII-pH demonstrated a reduced gastroesophageal reflux time, total number of liquid reflux episodes, and total number of reflux episodes (p < 0.001, p = 0.008, p = 0.009). Backflow prevention mechanism of laparoscopic Toupet fundoplication is thus considered to improve the overall LES function without elevating LESP.

Rethinking the laryngopharyngeal reflux treatment algorithm: Evaluating an alternate empiric dosing regimen and considering up-front, pH-impedance, and manometry testing to minimize cost in treating suspect laryngopharyngeal reflux disease.

PubMed

Carroll, Thomas L; Werner, Astrid; Nahikian, Kael; Dezube, Aaron; Roth, Douglas F

2017-10-01

Empiric proton pump inhibitor (PPI) trials for laryngopharyngeal reflux (LPR) are common. A majority of the patients respond to acid suppression. This work intends to evaluate once-daily, 40 mg omeprazole and once-nightly, 300 mg ranitidine (QD/QHS) dosing as an alternative regimen, and use this study's cohort to evaluate empiric regimens prescribed for LPR as compared to up-front testing with pH impedance multichannel intraluminal impedance (MII) with dual pH probes and high-resolution manometry (HRM) for potential cost minimization. Retrospective cohort review and cost minimization study. A chart review identified patients diagnosed with LPR. All subjects were treated sequentially and outcomes recorded. Initial QD/QHS dosing increased after 3 months to BID if no improvement and ultimately prescribed MII and HRM if they failed BID dosing. Decision tree diagrams were constructed to determine costs of two empiric regimens and up-front MII and HRM. Ninety-seven subjects met the criteria. Responders and nonresponders to empiric therapy were identified. Seventy-two subjects (74%) responded. Forty-eight (67% of responders and 49% of all) improved with QD/QHS dosing. Forty-nine (51%) subjects escalated to BID dosing. Twenty-four subjects (33% of responders and 25% of all) improved on BID therapy. Twenty-five subjects (26%) did not respond to acid suppression. Average weighted cost was $1,897.00 per patient for up-front testing, $3,033.00 for initial BID, and $3,366.00 for initial QD/QHS. An alternate QD/QHS regimen improved the majority who presented with presumed LPR. Cost estimates demonstrate that the QD/QHS regimen was more expensive than the initial BID high-dose PPI for 6 months. Overall per-patient cost appears less with up-front MII and HRM. 4. Laryngoscope, 127:S1-S13, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

Analysis of HIV Diversity in HIV-Infected Black Men Who Have Sex with Men (HPTN 061).

PubMed

Chen, Iris; Chau, Gordon; Wang, Jing; Clarke, William; Marzinke, Mark A; Cummings, Vanessa; Breaud, Autumn; Laeyendecker, Oliver; Fields, Sheldon D; Griffith, Sam; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

2016-01-01

HIV populations often diversify in response to selective pressures, such as the immune response and antiretroviral drug use. We analyzed HIV diversity in Black men who have sex with men who were enrolled in the HIV Prevention Trials Network 061 study. A high resolution melting (HRM) diversity assay was used to measure diversity in six regions of the HIV genome: two in gag, one in pol, and three in env. HIV diversity was analyzed for 146 men who were HIV infected at study enrollment, including three with acute infection and 13 with recent infection (identified using a multi-assay algorithm), and for 21 men who seroconverted during the study. HIV diversification was analyzed in a paired analysis for 62 HIV-infected men using plasma samples from the enrollment and 12-month (end of study) visits. Men with acute or recent infection at enrollment and seroconverters had lower median HRM scores (lower HIV diversity) than men with non-recent infection in all six regions analyzed. In univariate analyses, younger age, higher CD4 cell count, and HIV drug resistance were associated with lower median HRM scores in multiple regions; ARV drug detection was marginally associated with lower diversity in the pol region. In multivariate analysis, acute or recent infection (all six regions) and HIV drug resistance (both gag regions) were associated with lower median HRM scores. Diversification in the pol region over 12 months was greater for men with acute or recent infection, higher CD4 cell count, and lower HIV viral load at study enrollment. HIV diversity was significantly associated with duration of HIV infection, and lower gag diversity was observed in men who had HIV drug resistance. HIV pol diversification was more pronounced in men with acute or recent infection, higher CD4 cell count, and lower HIV viral load.

Normal values for high-resolution anorectal manometry in healthy women: effects of age and significance of rectoanal gradient.

PubMed

Noelting, Jessica; Ratuapli, Shiva K; Bharucha, Adil E; Harvey, Doris M; Ravi, Karthik; Zinsmeister, Alan R

2012-10-01

High-resolution manometry (HRM) is used to measure anal pressures in clinical practice but normal values have not been available. Although rectal evacuation is assessed by the rectoanal gradient during simulated evacuation, there is substantial overlap between healthy people and defecatory disorders, and the effects of age are unknown. We evaluated the effects of age on anorectal pressures and rectal balloon expulsion in healthy women. Anorectal pressures (HRM), rectal sensation, and balloon expulsion time (BET) were evaluated in 62 asymptomatic women ranging in age from 21 to 80 years (median age 44 years) without risk factors for anorectal trauma. In total, 30 women were aged <50 years. Age is associated with lower (r=-0.47, P<0.01) anal resting (63 (5) (≥50 years), 88 (3) (<50 years), mean (s.e.m.)) but not squeeze pressures; higher rectal pressure and rectoanal gradient during simulated evacuation (r=0.3, P<0.05); and a shorter (r=-0.4, P<0.01) rectal BET (17 (9) s (≥50 years) vs. 31 (10) s (<50 years)). Only 5 women had a prolonged (>60 s) rectal BET but 52 had higher anal than rectal pressures (i.e., negative gradient) during simulated evacuation. The gradient was more negative in younger (-41 (6) mm Hg) than older (-12 (6) mm Hg) women and negatively (r=-0.51, P<0.0001) correlated with rectal BET but only explained 16% of the variation in rectal BET. These observations provide normal values for anorectal pressures by HRM. Increasing age is associated with lower anal resting pressure, a more positive rectoanal gradient during simulated evacuation, and a shorter BET in asymptomatic women. Although the rectoanal gradient is negatively correlated with rectal BET, this gradient is negative even in a majority of asymptomatic women, undermining the utility of a negative gradient for diagnosing defecatory disorders by HRM.

Uncertainty in sap flow-based transpiration due to xylem properties

NASA Astrophysics Data System (ADS)

Looker, N. T.; Hu, J.; Martin, J. T.; Jencso, K. G.

2014-12-01

Transpiration, the evaporative loss of water from plants through their stomata, is a key component of the terrestrial water balance, influencing streamflow as well as regional convective systems. From a plant physiological perspective, transpiration is both a means of avoiding destructive leaf temperatures through evaporative cooling and a consequence of water loss through stomatal uptake of carbon dioxide. Despite its hydrologic and ecological significance, transpiration remains a notoriously challenging process to measure in heterogeneous landscapes. Sap flow methods, which estimate transpiration by tracking the velocity of a heat pulse emitted into the tree sap stream, have proven effective for relating transpiration dynamics to climatic variables. To scale sap flow-based transpiration from the measured domain (often <5 cm of tree cross-sectional area) to the whole-tree level, researchers generally assume constancy of scale factors (e.g., wood thermal diffusivity (k), radial and azimuthal distributions of sap velocity, and conducting sapwood area (As)) through time, across space, and within species. For the widely used heat-ratio sap flow method (HRM), we assessed the sensitivity of transpiration estimates to uncertainty in k (a function of wood moisture content and density) and As. A sensitivity analysis informed by distributions of wood moisture content, wood density and As sampled across a gradient of water availability indicates that uncertainty in these variables can impart substantial error when scaling sap flow measurements to the whole tree. For species with variable wood properties, the application of the HRM assuming a spatially constant k or As may systematically over- or underestimate whole-tree transpiration rates, resulting in compounded error in ecosystem-scale estimates of transpiration.

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates.

PubMed

Fang, H; Ohlsson, A-K; Ullberg, M; Ozenci, V

2012-11-01

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3 % (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis.

PubMed

Steer, P A; O'Rourke, D; Ghorashi, S A; Noormohammadi, A H

2011-05-01

Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. Twenty-six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high-resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. FAdV-8b and FAdV-11 were identified in 13 cases each. In one case, FAdV-1 was also identified. Cross-neutralisation was observed between the FAdV-11 field strain and the reference FAdV-2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV-8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV-11 field strain had the highest identity to FAdV-11 (93.2%) and FAdV-2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species. © 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.

The Chicago Classification 3.0 Results in More Normal Findings and Fewer Hypotensive Findings With No Difference in Other Diagnoses.

PubMed

Monrroy, H; Cisternas, D; Bilder, C; Ditaranto, A; Remes-Troche, J; Meixueiro, A; Zavala, M A; Serra, J; Marín, I; Ruiz de León, A; Pérez de la Serna, J; Hani, A; Leguizamo, A; Abrahao, L; Coello, R; Valdovinos, M A

2017-04-01

High-resolution manometry (HRM) is the preferred method for the evaluation of motility disorders. Recently, an update of the diagnostic criteria (Chicago 3.0) has been published. The aim of this study was to compare the performance criteria of Chicago version 2.0 (CC2.0) vs. 3.0 (CC3.0) in a cohort of healthy volunteers and symptomatic patients. HRM studies of asymptomatic and symptomatic individuals from several centers of Spain and Latin America were analyzed using both CC2.0 and CC3.0. The final diagnosis was grouped into hierarchical categories: obstruction (achalasia and gastro-esophageal junction obstruction), major disorders (distal esophageal spasm, absent peristalsis, and jackhammer), minor disorders (failed frequent peristalsis, weak peristalsis with small or large defects, ineffective esophageal motility, fragmented peristalsis, rapid contractile with normal latency and hypertensive peristalsis) and normal. The results were compared using McNemar's and Kappa tests. HRM was analyzed in 107 healthy volunteers (53.3% female; 18-69 years) and 400 symptomatic patients (58.5% female; 18-90 years). In healthy volunteers, using CC2.0 and CC3.0, obstructive disorders were diagnosed in 7.5% and 5.6%, respectively, major disorders in 1% and 2.8%, respectively, minor disorders in 25.2% and 15%, respectively, and normal in 66.4% and 76.6%, respectively. In symptomatic individuals, using CC2.0 and CC3.0, obstructive disorders were diagnosed in 11% and 11.3%, respectively, major disorders in 14% and 14%, respectively, minor disorders in 33.3% and 24.5%, respectively, and normal in 41.8% and 50.3%, respectively. In both groups of individuals, only an increase in normal and a decrease in minor findings using CC3.0 were statistically significant using McNemar's test. CC3.0 increases the number of normal studies when compared with CC2.0, essentially at the expense of fewer minor disorders, with no significant differences in major or obstructive disorders. As the relevance of minor disorders is questionable, our data suggest that CC3.0 increases the relevance of abnormal results.

Plasma metabolomics in adults with cystic fibrosis during a pulmonary exacerbation: a pilot randomized study of high-dose vitamin D3 administration

PubMed Central

Alvarez, Jessica A.; Chong, Elizabeth Y.; Walker, Douglas I.; Chandler, Joshua D.; Michalski, Ellen S.; Grossmann, Ruth E.; Uppal, Karan; Li, Shuzhao; Frediani, Jennifer K.; Tirouvanziam, Rabindra; Tran, ViLinh T.; Tangpricha, Vin; Jones, Dean P.; Ziegler, Thomas R.

2017-01-01

Background Cystic fibrosis (CF) is a chronic catabolic disease often requiring hospitalization for acute episodes of worsening pulmonary exacerbations. Limited data suggest that vitamin D may have beneficial clinical effects, but the impact of vitamin D on systemic metabolism in this setting is unknown. Objective We used high-resolution metabolomics (HRM) to assess the impact of baseline vitamin D status and high-dose vitamin D3 administration on systemic metabolism in adults with CF with an acute pulmonary exacerbation. Design Twenty-five hospitalized adults with CF were enrolled in a randomized trial of high-dose vitamin D3 (250,000 IU vitamin D3 bolus) versus placebo. Age-matched healthy subjects served as a reference group for baseline comparisons. Plasma was analyzed with liquid chromatography/ultra-high resolution mass spectrometry. Using recent HRM bioinformatics and metabolic pathway enrichment methods, we examined associations with baseline vitamin D status (sufficient vs deficient per serum 25-hydroxyvitamin D concentrations) and the 7-day response to vitamin D3 supplementation. Results Several amino acids and lipid metabolites differed between CF and healthy control subjects, indicative of an overall catabolic state. In CF subjects, 343 metabolites differed (P<0.05) by baseline vitamin D status and were enriched within 7 metabolic pathways including fatty acid, amino acid, and carbohydrate metabolism. A total of 316 metabolites, which showed enrichment for 15 metabolic pathways--predominantly representing amino acid pathways-- differed between the vitamin D3- and placebo-treated CF subjects over time (P<0.05). In the placebo group, several tricarboxylic acid cycle intermediates increased while several amino acid-related metabolites decreased; in contrast, little change in these metabolites occurred with vitamin D3 treatment. Conclusions Numerous metabolic pathways detected by HRM varied in association with vitamin D status and high-dose vitamin D3 supplementation in adults with CF experiencing a pulmonary exacerbation. Overall, these pilot data suggest an anti-catabolic effect of high-dose vitamin D3 in this clinical setting. PMID:28403943

High-Resolution Manometry Evaluation of Pressures at the Pharyngo-upper Esophageal Area in Patients with Oropharyngeal Dysphagia Due to Vagal Paralysis.

PubMed

Pinna, Bruno Rezende; Herbella, Fernando A M; de Biase, Noemi; Vaiano, Thays C G; Patti, Marco G

2017-10-01

The motility of the pharynx, upper esophageal sphincter (UES), and proximal esophagus in patients with oropharyngeal dysphagia is still not entirely understood. High-resolution manometry (HRM) was recently added to the armamentarium for the study of this area. This study aims to describe HRM findings in patients with vagal paralysis. Sixteen patients (mean age 54 years, 69% females) with oropharyngeal dysphagia due to unilateral vagal paralysis were prospectively studied. All patients underwent HRM. Motility of the UES and at the topography of the velopharynx and epiglottis were recorded. (1) UES relaxation is compromised in a minority of patients, (2) epiglottis pressure does not follow a specific pattern, (3) vellum is hypotonic in half of the patients, (4) dysphagia is related to a low pharyngeal pressure, not to a flow obstruction at the level of the UES, and (5) aspiration is related to low pressures at the level of the UES and epiglottis and higher pressures at the level of the vellum. Pharyngeal motility is significantly impaired in patients with oropharyngeal dysphagia and unilateral vagal paralysis. In half of the cases, UES resting pressure is preserved due to unilateral innervation and relaxation is normal in most patients. Dysphagia therapy in these patients must be directed toward improvement in the oropharyngeal motility not at the UES.

Hydrogen-rich saline attenuates vascular smooth muscle cell proliferation and neointimal hyperplasia by inhibiting reactive oxygen species production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways.

PubMed

Chen, Yali; Jiang, Jinyao; Miao, Huibing; Chen, Xingjuan; Sun, Xuejun; Li, Yongjun

2013-03-01

Hydrogen-rich saline has been reported to prevent neointimal hyperplasia induced by carotid balloon injury. The purpose of the present study was to further investigate the molecular mechanisms underlying this phenomenon. Daily injection of a hydrogen-rich saline solution (HRSS) in rats was employed to study the effect of hydrogen on balloon injury-induced neointimal hyperplasia and the neointima/media ratio was assessed. HRSS significantly decreased the neointima area and neointima/media ratio in a dose-dependent manner. In vitro effects of hydrogen on fetal bovine serum (FBS)-induced vascular smooth muscle cell (VSMC) proliferation were also investigated. Hydrogen-rich medium (HRM) inhibited rat VSMC proliferation and migration induced by 10% FBS. FBS-induced reactive oxygen species (ROS) production and activation of intracellular Ras, MEK1/2, ERK1/2, proliferative cell nuclear antigen (PCNA), Akt were significantly inhibited by HRM. In addition, HRM blocked FBS-induced progression from the G0/G1 to the S-phase and increased the apoptosis rate of VSMCs. These results showed that hydrogen-rich saline was able to attenuate FBS-induced VSMC proliferation and neointimal hyperplasia by inhibiting ROS production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways. Thus, HRSS may have potential therapeutic relevance for the prevention of human restenosis.

In vitro antioxidant profiling of seabuckthorn varieties and their adaptogenic response to high altitude-induced stress

NASA Astrophysics Data System (ADS)

Sharma, Priyanka; Suryakumar, Geetha; Singh, Virendra; Misra, Kshipra; Singh, Shashi Bala

2015-08-01

In the past few years, seabuckthorn plants have gained special attention due to their ability to grow in the harshest of the environment. This adaptability may be contributed by various antioxidants present in the plants besides other morphological adaptation. As in vivo studies cannot be justified without in vitro studies, the present investigation carried out evaluation of both in vitro and in vivo antioxidant potentials of aqueous and alcoholic extracts of the leaves of Hippophae salicifolia (HS) and Hippophae rhamnoides mongolica (HRM) in comparison with Hippophae rhamnoides turkestanica (HRT). The results had clearly depicted that in vitro antioxidant potential of the extracts was responsible for the in vivo adaptogenic performance in animals during cold and hypoxia exposure under restraint stress. Total phenolic content (TPC), total flavonoid content (TFC), total protein content, and antioxidant potential were determined. For adaptogenic studies, rats with oral drug supplementation were exposed to Cold-hypoxia-restraint (C-H-R) stresses-induced hypothermia, as a measure of endurance. Aqueous extracts of HS showed maximum (99 %) resistance compared to HRT (81 %) and HRM (29 %). The levels of biochemical parameters such as malondialdehyde (MDA), reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione (GSH/GSSG), and catalase (CAT) in blood samples also revealed that the aqueous leaf extract of HS has better antioxidant and adaptogenic potential compared to HRM.

High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

PubMed Central

Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

PubMed

Ngoi, Soo Tein; Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

Dor Vs Toupet Fundoplication After Laparoscopic Heller Myotomy: Long-Term Randomized Controlled Trial Evaluated by High-Resolution Manometry.

PubMed

Torres-Villalobos, Gonzalo; Coss-Adame, Enrique; Furuzawa-Carballeda, Janette; Romero-Hernández, Fernanda; Blancas-Breña, Blanca; Torres-Landa, Samuel; Palacios-Ramírez, Axel; Alejandro-Medrano, Edgar; Hernández-Ávila, Axel; Flores-Najera, Athenea; Ávila Escobedo, Lourdes Margarita; Ramírez Angulo, Cecilia; Rodríguez-Garcés, Angélica; Valdovinos, Miguel Ángel

2018-01-01

Laparoscopic Heller myotomy (LHM) with partial fundoplication is an effective treatment for achalasia. However, the type of fundoplication is still a subject of debate. The aim of the study is to identify which partial fundoplication leads to better control of acid exposure, manometric parameters, and symptoms scores. A randomized controlled trial was performed to compare Dor vs Toupet fundoplication after LHM. The preoperative diagnosis was made by high-resolution manometry (HRM), upper endoscopy, and barium esophagogram. Preoperative and postoperative symptoms were evaluated with Eckardt, GERD-HRQL, and EAT-10 questionnaires. Seventy-three patients were randomized, 38 underwent Dor and 35 Toupet. Baseline characteristics were similar between groups. Postoperative HRM showed that the integrated relaxation pressure (IRP) and basal lower esophageal sphincter (LES) pressure were similar at 6 and 24 months. The number of patients with abnormal acid exposure was significantly lower for Dor (6.9%) than that of Toupet (34.0%) at 6 months, but it was not different at 12 or 24 months. No differences were found in postoperative symptom scores at 1, 6, or 24 months. There were no differences in symptom scores or HRM between fundoplications in the long term. A higher percentage of abnormal 24-h pH test were found for the Toupet group, with no difference in the long term.

Hard breakup of the deuteron into two Δ -isobars

NASA Astrophysics Data System (ADS)

Granados, Carlos; Sargsian, Misak

2011-04-01

Photodisintegration of the deuteron into two Δ-isobars at large center of mass angles is studied within the QCD hard rescattering model (HRM). According to the HRM, the reaction proceeds in three main steps: the photon knocks the quark from one of the nucleons in the deuteron; the struck quark rescatters off a quark from the other nucleon sharing the high energy of the photon; then the energetic quarks recombine into two outgoing baryons emerging at large transverse momenta. Within the HRM, the cross section is expressed through the amplitude of pn --> ΔΔ scattering which we evaluated based on the quark-interchange model of hard hadronic scattering. We predict that the cross section of the deuteron breakup to Δ++Δ- is 4-5 times larger than that of the breakup to the Δ+Δ0 channel. Also, the angular distributions for these two channels are markedly different. These can be compared with the predictions based on the assumption that two hard Δ-isobars are the result of the disintegration of initial ΔΔ components of the deuteron wave function. In this case, the angular distributions and cross sections of the breakup in both Δ++Δ- and Δ+Δ0 channels are expected to be similar. This work was supported by U.S. Department of Energy Grant under contract DE-FG02-01ER41172, and by the FIU DEA program.

Hard breakup of the deuteron into two Δ isobars

NASA Astrophysics Data System (ADS)

Granados, Carlos G.; Sargsian, Misak M.

2011-05-01

We study high-energy photodisintegration of the deuteron into two Δ isobars at large center of mass angles within the QCD hard rescattering model (HRM). According to the HRM, the process develops in three main steps: the photon knocks a quark from one of the nucleons in the deuteron; the struck quark rescatters off a quark from the other nucleon sharing the high energy of the photon; then the energetic quarks recombine into two outgoing baryons which have large transverse momenta. Within the HRM, the cross section is expressed through the amplitude of pn→ΔΔ scattering which we evaluated based on the quark-interchange model of hard hadronic scattering. Calculations show that the angular distribution and the strength of the photodisintegration is mainly determined by the properties of the pn→ΔΔ scattering. We predict that the cross section of the deuteron breakup to Δ++Δ- is 4-5 times larger than that of the breakup to the Δ+Δ0 channel. Also, the angular distributions for these two channels are markedly different. These can be compared with the predictions based on the assumption that two hard Δ isobars are the result of the disintegration of the preexisting ΔΔ components of the deuteron wave function. In this case, one expects the angular distributions and cross sections of the breakup in both Δ++Δ- and Δ+Δ0 channels to be similar.

A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE specimens.

PubMed

Huebner, Claudia; Weber, Remeny; Lloydd, Richard

2017-12-01

Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

PubMed

di Rienzo, Valentina; Bubici, Giovanni; Montemurro, Cinzia; Cillo, Fabrizio

2018-01-01

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

Advances in Management of Esophageal Motility Disorders.

PubMed

Kahrilas, Peter J; Bredenoord, Albert J; Carlson, Dustin A; Pandolfino, John E

2018-04-24

The widespread adoption of high-resolution manometry (HRM) has led to a restructuring in the classification of esophageal motility disorder classification summarized in the Chicago Classification, currently in version 3.0. It has become apparent that the cardinal feature of achalasia, impaired lower esophageal sphincter relaxation, can occur in several disease phenotypes: without peristalsis, with premature (spastic) distal esophageal contractions, with panesophageal pressurization, or even with preserved peristalsis. Furthermore, despite these advances in diagnostics, no single manometric pattern is perfectly sensitive or specific for idiopathic achalasia and complimentary assessments with provocative maneuvers during HRM or interrogating the esophagogastric junction with the functional luminal imaging probe during endoscopy can be useful in clarifying equivocal or inexplicable HRM findings. Using these tools, we have come to conceptualize esophageal motility disorders as characterized by obstructive physiology at the esophagogastric junction, smooth muscle esophagus, or both. Recognizing obstructive physiology as a primary target of therapy has become particularly relevant with the development of a minimally invasive technique for performing a calibrated myotomy of the esophageal circular muscle, the POEM procedure. Now and going forward, optimal management is to render treatment in a phenotype-specific manner: e.g. POEM calibrated to patient-specific physiology for spastic achalasia and spastic disorders of the smooth muscle esophagus, more conservative strategies (pneumatic dilation) for the disorders limited to the sphincter. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

G894T endothelial nitric oxide synthase polymorphism and ischemic stroke in Morocco

PubMed Central

Diakite, Brehima; Hamzi, Khalil; Slassi, Ilham; EL Yahyaoui, Mohammed; EL Alaoui, Moulay M.F.; Habbal, Rachida; Sellama, Nadifi

2014-01-01

Nitric oxide plays a major role in the regulation of cerebral blood flow and loss of its function leads to alteration of the vascular relaxation given its central role in the physiology of the vascular system. G894T eNOS polymorphism could have adverse effects on the expression and activity of endothelial nitric oxide synthase, which can result in functional impairment of the endothelium and contribute to the development of ischemic stroke in the different models of transmission. In this study, genotyping with PCR-RFLP and HRM (high resolution melting) methods were conducted on 165 ischemic stroke patients as well as 182 controls. The goal here was to compare genotyping with PCR-RLFP primer sequences of eNOS gene (size < 300 bp) to HRM. Our data suggests a statistically significant association between G894T eNOS polymorphism and ischemic stroke in recessive, dominant and additive models with P < 0.05 and odds ratio of 2.68 (1.08–6.70), 1.78 (1.16–2.73), and 1.71 (1.21–2.43) respectively. In sum, although the sample size is relatively small, it suggests that G894T eNOS polymorphism could be a potentially important genetic marker of ischemic stroke in the Moroccan population. Future studies should be conducted in this direction taking into consideration the functional activity of eNOS. PMID:25606419

High resolution probabilistic precipitation forecast over Spain combining the statistical downscaling tool PROMETEO and the AEMET short range EPS system (AEMET/SREPS)

NASA Astrophysics Data System (ADS)

Cofino, A. S.; Santos, C.; Garcia-Moya, J. A.; Gutierrez, J. M.; Orfila, B.

2009-04-01

The Short-Range Ensemble Prediction System (SREPS) is a multi-LAM (UM, HIRLAM, MM5, LM and HRM) multi analysis/boundary conditions (ECMWF, UKMetOffice, DWD and GFS) run twice a day by AEMET (72 hours lead time) over a European domain, with a total of 5 (LAMs) x 4 (GCMs) = 20 members. One of the main goals of this project is analyzing the impact of models and boundary conditions in the short-range high-resolution forecasted precipitation. A previous validation of this method has been done considering a set of climate networks in Spain, France and Germany, by interpolating the prediction to the gauge locations (SREPS, 2008). In this work we compare these results with those obtained by using a statistical downscaling method to post-process the global predictions, obtaining an "advanced interpolation" for the local precipitation using climate network precipitation observations. In particular, we apply the PROMETEO downscaling system based on analogs and compare the SREPS ensemble of 20 members with the PROMETEO statistical ensemble of 5 (analog ensemble) x 4 (GCMs) = 20 members. Moreover, we will also compare the performance of a combined approach post-processing the SREPS outputs using the PROMETEO system. References: SREPS 2008. 2008 EWGLAM-SRNWP Meeting (http://www.aemet.es/documentos/va/divulgacion/conferencias/prediccion/Ewglam/PRED_CSantos.pdf)

Clinical and manometric characteristics of patients with Parkinson's disease and esophageal symptoms.

PubMed

Su, A; Gandhy, R; Barlow, C; Triadafilopoulos, G

2017-04-01

Dysphagia is a common problem in patients with Parkinson's disease (PD); its etiology is multifactorial and its management is challenging. In this retrospective cohort analysis using prospectively collected data, we aimed to objectively characterize dysphagia and/or other esophageal symptoms in patients with PD, assess the prevalence of outflow obstruction as well as major or minor disorders of esophageal peristalsis leading to impaired esophageal clearance and highlight objective parameters that can help in the current management algorithm. Thirty-three consecutive patients with PD presenting with dysphagia, odynophagia, heartburn, regurgitation, chest pain, and weight loss underwent clinical and functional evaluation by high-resolution manometry (HRM). Esophagogastric junction (EGJ) outflow obstruction and major as well as minor disorders of peristalsis were then assessed using the Chicago classification (v3). Thirty-three PD patients with esophageal symptoms were enrolled in the study; 12 of them reported weight loss that was considered as potentially reflecting underlying esophageal dysfunction. The median age of the patients was 70 years (range: 53-89 years), 24 (75%) were men. The majority (62%) experienced dysphagia, likely contributing to weight loss in 41% of patients. Odynophagia was rare (6%) while GER symptoms, such as heartburn, regurgitation, and chest pain were noted in 37%, 31%, and 28% of patients, respectively. Using the hierarchy of the Chicago classification, 12 patients (39%) exhibited EGJ outflow obstruction, 16 (48%) diffuse esophageal spasm (DES), 18 (55%), ineffective esophageal peristalsis (IEM), 16 (48%) fragmented peristalsis, and only 2 patients (6%) had normal HRM tracings. There were no patients with HRM features of achalasia. Dysphagia is common in patients with PD and is associated with a high prevalence of underlying motility disturbances as identified by HRM. The exact impact of these motility abnormalities on symptom induction and their role in influencing clinical management are unclear and will require further study. © The Authors 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Elevated intrabolus pressure identifies obstructive processes when integrated relaxation pressure is normal on esophageal high-resolution manometry.

PubMed

Quader, Farhan; Reddy, Chanakyaram; Patel, Amit; Gyawali, C Prakash

2017-07-01

Elevated integrated relaxation pressure (IRP) on esophageal high-resolution manometry (HRM) identifies obstructive processes at the esophagogastric junction (EGJ). Our aim was to determine whether intrabolus pressure (IBP) can identify structural EGJ processes when IRP is normal. In this observational cohort study, adult patients with dysphagia and undergoing HRM were evaluated for endoscopic evidence of structural EGJ processes (strictures, rings, hiatus hernia) in the setting of normal IRP. HRM metrics [IRP, distal contractile integral (DCI), distal latency (DL), IBP, and EGJ contractile integral (EGJ-CI)] were compared among 74 patients with structural EGJ findings (62.8 ± 1.6 yr, 67.6% women), 27 patients with normal EGD (52.9 ± 3.2 yr, 70.3% women), and 21 healthy controls (27.6 ± 0.6 yr, 52.4% women). Findings were validated in 85 consecutive symptomatic patients to address clinical utility. In the primary cohort, mean IBP (18.4 ± 0.9 mmHg) was higher with structural EGJ findings compared with dysphagia with normal EGD (13.5 ± 1.1 mmHg, P = 0.002) and healthy controls (10.9 ± 0.9 mmHg, P < 0.001). However, mean IRP, DCI, DL, and EGJ-CI were similar across groups ( P > 0.05 for each comparison). During multiple rapid swallows, IBP remained higher in the structural findings group compared with controls ( P = 0.02). Similar analysis of the prospective validation cohort confirmed IBP elevation in structural EGJ processes, but correlation with dysphagia could not be demonstrated. We conclude that elevated IBP predicts the presence of structural EGJ processes even when IRP is normal, but correlation with dysphagia is suboptimal. NEW & NOTEWORTHY Integrated relaxation pressure (IRP) above the upper limit of normal defines esophageal outflow obstruction using high-resolution manometry. In patients with normal IRP, elevated intrabolus pressure (IBP) can be a surrogate marker for a structural restrictive or obstructive process at the esophagogastric junction (EGJ). This has the potential to augment the clinical value of esophageal HRM by raising suspicion for a structural EGJ process when IBP is elevated.

QCD structure of nuclear interactions

NASA Astrophysics Data System (ADS)

Granados, Carlos G.

The research presented in this dissertation investigated selected processes involving baryons and nuclei in hard scattering reactions. These processes are characterized by the production of particles with large energies and transverse momenta. Through these processes, this work explored both, the constituent (quark) structure of baryons (specifically nucleons and Delta-Isobars), and the mechanisms through which the interactions between these constituents ultimately control the selected reactions. The first of such reactions is the hard nucleon-nucleon elastic scattering, which was studied here considering the quark exchange between the nucleons to be the dominant mechanism of interaction in the constituent picture. In particular, it was found that an angular asymmetry exhibited by proton-neutron elastic scattering data is explained within this framework if a quark-diquark picture dominates the nucleon's structure instead of a more traditional SU(6) three quarks picture. The latter yields an asymmetry around 90o center of mass scattering with a sign opposite to what is experimentally observed. The second process is the hard breakup by a photon of a nucleon-nucleon system in light nuclei. Proton-proton (pp) and proton-neutron (pn) breakup in 3He, and DeltaDelta-isobars production in deuteron breakup were analyzed in the hard rescattering model (HRM), which in conjunction with the quark interchange mechanism provides a Quantum Chromodynamics (QCD) description of the reaction. Through the HRM, cross sections for both channels in 3He photodisintegration were computed without the need of a fitting parameter. The results presented here for pp breakup show excellent agreement with recent experimental data. In DeltaDelta-isobars production in deuteron breakup, HRM angular distributions for the two DeltaDelta channels were compared to the pn channel and to each other. An important prediction fromthis study is that the Delta++Delta- channel consistently dominates Delta+Delta0, which is in contrast with models that unlike the HRM consider a DeltaDelta system in the initial state of the interaction. For such models both channels should have the same strength. These results are important in developing a QCD description of the atomic nucleus.

Application of stochastic approach based on Monte Carlo (MC) simulation for life cycle inventory (LCI) to the steel process chain: case study.

PubMed

Bieda, Bogusław

2014-05-15

The purpose of the paper is to present the results of application of stochastic approach based on Monte Carlo (MC) simulation for life cycle inventory (LCI) data of Mittal Steel Poland (MSP) complex in Kraków, Poland. In order to assess the uncertainty, the software CrystalBall® (CB), which is associated with Microsoft® Excel spreadsheet model, is used. The framework of the study was originally carried out for 2005. The total production of steel, coke, pig iron, sinter, slabs from continuous steel casting (CSC), sheets from hot rolling mill (HRM) and blast furnace gas, collected in 2005 from MSP was analyzed and used for MC simulation of the LCI model. In order to describe random nature of all main products used in this study, normal distribution has been applied. The results of the simulation (10,000 trials) performed with the use of CB consist of frequency charts and statistical reports. The results of this study can be used as the first step in performing a full LCA analysis in the steel industry. Further, it is concluded that the stochastic approach is a powerful method for quantifying parameter uncertainty in LCA/LCI studies and it can be applied to any steel industry. The results obtained from this study can help practitioners and decision-makers in the steel production management. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

PubMed

Fidler, Gabor; Kocsube, Sandor; Leiter, Eva; Biro, Sandor; Paholcsek, Melinda

2017-08-01

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

PubMed Central

2011-01-01

Background The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. Methods We implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment. Results We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. Conclusions Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms. PMID:21569354

High density bit transition requirements versus the effects on BCH error correcting code. [bit synchronization

NASA Technical Reports Server (NTRS)

Ingels, F. M.; Schoggen, W. O.

1982-01-01

The design to achieve the required bit transition density for the Space Shuttle high rate multiplexes (HRM) data stream of the Space Laboratory Vehicle is reviewed. It contained a recommended circuit approach, specified the pseudo random (PN) sequence to be used and detailed the properties of the sequence. Calculations showing the probability of failing to meet the required transition density were included. A computer simulation of the data stream and PN cover sequence was provided. All worst case situations were simulated and the bit transition density exceeded that required. The Preliminary Design Review and the critical Design Review are documented. The Cover Sequence Generator (CSG) Encoder/Decoder design was constructed and demonstrated. The demonstrations were successful. All HRM and HRDM units incorporate the CSG encoder or CSG decoder as appropriate.

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer.

PubMed

Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi

2012-06-15

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of Head Rotation and Head Tilt on Pharyngeal Pressure Events Using High Resolution Manometry.

PubMed

Kim, Cheol Ki; Ryu, Ju Seok; Song, Sun Hong; Koo, Jung Hoi; Lee, Kyung Duck; Park, Hee Sun; Oh, Yoongul; Min, Kyunghoon

2015-06-01

To observe changes in pharyngeal pressure during the swallowing process according to postures in normal individuals using high-resolution manometry (HRM). Ten healthy volunteers drank 5 mL of water twice while sitting in a neutral posture. Thereafter, they drank the same amount of water twice in the head rotation and head tilting postures. The pressure and time during the deglutition process for each posture were measured with HRM. The data obtained for these two postures were compared with those obtained from the neutral posture. The maximum pressure, area, rise time, and duration in velopharynx (VP) and tongue base (TB) were not affected by changes in posture. In comparison, the maximum pressure and the pre-upper esophageal sphincter (UES) maximum pressure of the lower pharynx in the counter-catheter head rotation posture were lower than those in the neutral posture. The lower pharynx pressure in the catheter head tilting posture was higher than that in the counter-catheter head tilting. The changes in the VP peak and epiglottis, VP and TB peaks, and the VP onset and post-UES time intervals were significant in head tilting and head rotation toward the catheter postures, as compared with neutral posture. The pharyngeal pressure and time parameter analysis using HRM determined the availability of head rotation as a compensatory technique for safe swallowing. Tilting the head smoothes the progress of food by increasing the pressure in the pharynx.

The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

PubMed

Jegal, Jonghwan; Chung, Ki Wung; Chung, Hae Young; Jeong, Eun Ju; Yang, Min Hye

2017-01-01

In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-β-D-xylopyranoside and isoscutellarein-7-O-β-D-xylopyranoside as main components. Hypolaetin-7-O-β-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

Courseware Review.

ERIC Educational Resources Information Center

Risley, John, Ed.

1988-01-01

Compares the features of the sonic rangers available from HRM Software, MICROMEASUREMENTS, NAGAWTIS Software Research, and PASCO Scientific for demonstrations and experiments in mechanics. Presents the advantages of the sonic rangers and the typical graphics displayed by each software package. (YP)

Airline Maintenance Manpower Optimization from the De Novo Perspective

NASA Astrophysics Data System (ADS)

Liou, James J. H.; Tzeng, Gwo-Hshiung

Human resource management (HRM) is an important issue for today’s competitive airline marketing. In this paper, we discuss a multi-objective model designed from the De Novo perspective to help airlines optimize their maintenance manpower portfolio. The effectiveness of the model and solution algorithm is demonstrated in an empirical study of the optimization of the human resources needed for airline line maintenance. Both De Novo and traditional multiple objective programming (MOP) methods are analyzed. A comparison of the results with those of traditional MOP indicates that the proposed model and solution algorithm does provide better performance and an improved human resource portfolio.

High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

PubMed

Girault, G; Wattiau, P; Saqib, M; Martin, B; Vorimore, F; Singha, H; Engelsma, M; Roest, H J; Spicic, S; Grunow, R; Vicari, N; De Keersmaecker, S C J; Roosens, N H C; Fabbi, M; Tripathi, B N; Zientara, S; Madani, N; Laroucau, K

2018-05-08

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional restoration of the esophagus after peroral endoscopic myotomy for achalasia

PubMed Central

Huh, Cheal Wung; Youn, Young Hoon; Chung, Hyunsoo; Lee, Yong Chan; Park, Hyojin

2017-01-01

Purpose Peroral endoscopic myotomy (POEM) is a new efficacious treatment option for achalasia. We propose to define “esophageal remodeling” as the functional restoration of the esophagus that involves decreased lower esophageal sphincter (LES) pressure, recovery of esophageal body peristalsis, and reduction of luminal diameter. The aim of this study was to investigate “esophageal remodeling” after POEM for achalasia. Materials and methods We analyzed data from a prospectively collected database of POEM subjects, which included preoperative and 2-month postoperative Eckardt symptom scores, and results from esophageal high resolution manometry (HRM) and barium esophagogram (BE). We recruited 23 patients (13 male; mean age: 53.9 years) whose preoperative and postoperative HRM and BE results were available, from among 30 patients with achalasia who underwent POEM at two institutions between July 2013 and December 2015. Results All patients achieved clinical treatment success (Eckardt score≤3). Partial recovery of esophageal body peristalsis was noted in 1/5 patients with type I (20%), 6/11 with type II (54.5%), and 7/7 with type III (100%) achalasia after POEM. Pan-esophageal pressurization disappeared after POEM in 10/11 type II achalasia patients. The average diameter of the esophageal body after POEM was significantly decreased in all types of achalasia. Conclusion POEM provided excellent clinical symptomatic relief and esophageal remodeling in terms of restoration of peristalsis and reduction in diameter of the esophageal body, especially in patients with type III achalasia. PMID:28542509

Incidence and molecular characterization of Glucose-6-Phosphate Dehydrogenase deficiency among neonates for newborn screening in Chaozhou, China.

PubMed

Yang, H; Wang, Q; Zheng, L; Zhan, X-F; Lin, M; Lin, F; Tong, X; Luo, Z-Y; Huang, Y; Yang, L-Y

2015-06-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is highly prevalent in southern China. The aim of this study is to assess the extent of this disease in Chinese neonates and determine its molecular characteristics using a novel molecular screening method. A total of 2500 neonates were routinely screened for G6PD deficiency using a modified fluorescent spot test (FST). PCR-high-resolution melting (HRM) analysis was then used for the molecular assay. The overall incidence of G6PD deficiency was 2.68% in our study cohort. Frequency in male population was 3.22% (44 neonates of 1365 male neonates), and in female population was 2.03% (23 neonates of 1135 female neonates). Of the 67 newborns suspected to be G6PD deficient based on FST (44 males, 23 females), 58 of 67 (87%) were detected with gene alterations. Seven kinds of mutations [c.95A>G, c.392G>T, c.493A>G, c.871G>A, c.1360C>T, c.1376G>T, and c.1388G>A] were identified by HRM analysis. Routine newborn screening in Chaozhou, China with a relatively high prevalence of G6PD deficiency is justified and meets the World Health Organization recommendation. The usage of molecular diagnosis can favor the detection of heterozygotes which can be a supplement to regular newborn screening and useful for premarital and prenatal diagnosis for G6PD deficiency. © 2014 John Wiley & Sons Ltd.