ResearchPad - melting https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[How global DNA unwinding causes non-uniform stress distribution and melting of DNA]]> https://www.researchpad.co/article/elastic_article_14712 DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.

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<![CDATA[Micro- and mesozooplankton successions in an Antarctic coastal environment during a warm year]]> https://www.researchpad.co/article/elastic_article_14619 The rapid increase in atmospheric temperature detected in the last decades in the Western Antarctic Peninsula was accompanied by a strong glacier retreat and an increase in production of melting water, as well as changes in the sea-ice dynamic. The objective of this study was to analyze the succession of micro- and mesozooplankton during a warm annual cycle (December 2010-December 2011) in an Antarctic coastal environment (Potter Cove). The biomass of zooplankton body size classes was used to predict predator-prey size relationships (i.e., to test bottom-up/top-down control effects) using a Multiple Linear Regression Analysis. The micro- and mesozooplanktonic successions were graphically analyzed to detect the influence of environmental periods (defined by the degree of glacial melting, sea-ice freezing and sea-ice melting) on coupling/uncoupling planktonic biomass curves associated to possible predator-prey size relationship scenarios. At the beginning of the glacial melting, medium and large mesozooplankton (calanoid copepods, Euphausia superba, and Salpa thompsoni) exert a top-down control on Chl-a and microzooplankton. Stratification of the water column benefitted the availability of adequate food-size (Chl-a <20) for large microzooplankton (tintinnids) development observed during fall. High abundance of omnivores mesozooplankton (Oithona similis and furcilia of E. superba) during sea-ice freezing periods would be due to the presence of available heterotrophic food under or within the sea ice. Finally, the increase in microzooplankton abundance in the middle of spring, when sea-ice melting starts, corresponded to small and medium dinoflagellates and ciliates species, which were possibly part of the biota of sea ice. If glacier retreat continues and the duration and thickness of the sea ice layer fluctuates as predicted by climate models, our results predict a future scenario regarding the zooplankton succession in Antarctic coastal environments.

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<![CDATA[Pressure‐Induced Coordination Changes in a Pyrolitic Silicate Melt From Ab Initio Molecular Dynamics Simulations]]> https://www.researchpad.co/article/Nb158e38a-7b2f-4be5-a947-08f65db4b3f1

Abstract

With ab initio molecular dynamics simulations on a Na‐, Ca‐, Fe‐, Mg‐, and Al‐bearing silicate melt of pyrolite composition, we examine the detailed changes in elemental coordination as a function of pressure and temperature. We consider the average coordination as well as the proportion and distribution of coordination environments at pressures and temperatures encompassing the conditions at which molten silicates may exist in present‐day Earth and those of the Early Earth's magma ocean. At ambient pressure and 2,000 K, we find that the average coordination of cations with respect to oxygen is 4.0 for Si‐O, 4.0 for Al‐O, 3.7 for Fe‐O, 4.6 for Mg‐O, 5.9 for Na‐O, and 6.2 for Ca‐O. Although the coordination for iron with respect to oxygen may be underestimated, the coordination number for all other cations are consistent with experiments. By 15 GPa (2,000 K), the average coordination for Si‐O remains at 4.0 but increases to 4.1 for Al‐O, 4.2 for Fe‐O, 4.9 for Mg‐O, 8.0 for Na‐O, and 6.8 for Ca‐O. The coordination environment for Na‐O remains approximately constant up to core‐mantle boundary conditions (135 GPa and 4000 K) but increases to about 6 for Si‐O, 6.5 for Al‐O, 6.5 for Fe‐O, 8 for Mg‐O, and 9.5 for Ca‐O. We discuss our results in the context of the metal‐silicate partitioning behavior of siderophile elements and the viscosity changes of silicate melts at upper mantle conditions. Our results have implications for melt properties, such as viscosity, transport coefficients, thermal conductivities, and electrical conductivities, and will help interpret experimental results on silicate glasses.

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<![CDATA[Screening of brain-derived neurotrophic factor (BDNF) single nucleotide polymorphisms and plasma BDNF levels among Malaysian major depressive disorder patients]]> https://www.researchpad.co/article/5c536c08d5eed0c484a497c8

Background

Brain-derived neurotrophic factor (BDNF) is a neurotrophin found in abundance in brain regions such as the hippocampus, cortex, cerebellum and basal forebrain. It has been associated with the risk of susceptibility to major depressive disorder (MDD). This study aimed to determine the association of three BDNF variants (rs6265, rs1048218 and rs1048220) with Malaysian MDD patients.

Methods

The correlation of these variants to the plasma BDNF level among Malaysian MDD patients was assessed. A total of 300 cases and 300 matched controls recruited from four public hospitals within the Klang Valley of Selangor State, Malaysia and matched for age, sex and ethnicity were screened for BDNF rs6265, rs1048218 and rs1048220 using high resolution melting (HRM).

Findings

BDNF rs1048218 and BDNF rs1048220 were monomorphic and were excluded from further analysis. The distribution of the alleles and genotypes for BDNF rs6265 was in Hardy-Weinberg equilibrium for the controls (p = 0.13) but was in Hardy Weinberg disequilibrium for the cases (p = 0.011). Findings from this study indicated that having BDNF rs6265 in the Malaysian population increase the odds of developing MDD by 2.05 folds (95% CI = 1.48–3.65). Plasma from 206 cases and 206 controls were randomly selected to measure the BDNF level using enzyme-linked immunosorbent assay (ELISA). A significant decrease in the plasma BDNF level of the cases as compared to controls (p<0.0001) was observed. However, there was no evidence of the effect of the rs6265 genotypes on the BDNF level indicating a possible role of other factors in modulating the BDNF level that warrants further investigation.

Conclusion

The study indicated that having the BDNF rs6265 allele (A) increase the risk of developing MDD in the Malaysian population suggesting a possible role of BDNF in the etiology of the disorder.

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<![CDATA[A method for the detection and characterization of technology fronts: Analysis of the dynamics of technological change in 3D printing technology]]> https://www.researchpad.co/article/5c3d0112d5eed0c4840380e9

This paper presents a method for the identification of the “technology fronts”—core technological solutions—underlying a certain broad technology, and the characterization of their change dynamics. We propose an approach based on the Latent Dirichlet Allocation (LDA) model combined with patent data analysis and text mining techniques for the identification and dynamic characterization of the main fronts where actual technological solutions are put into practice. 3D printing technology has been selected to put our method into practice for its market emergence and multidisciplinarity. The results show two highly relevant and specialized fronts strongly related with mechanical design that evolve gradually, in our opinion acting as enabling technologies. On the other side, we detected three fronts undergoing significant changes, namely layer-by-layer multimaterial manufacturing, data processing and stereolithograpy techniques. Laser and electron-beam based technologies take shape in the latter years and show signs of becoming enabling technologies in the future. The technology fronts and data revealed by our method have been convincing to experts and coincident with many technology trends already pointed out in technical reports and scientific literature.

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<![CDATA[Experimental study on frost-formation characteristics on cold surface of arched copper sample]]> https://www.researchpad.co/article/5c1966f4d5eed0c484b536c4

The present work investigates the process of frosting formation on arched copper samples with different surface temperatures, calculated the thickness of the frost layer by using the scale method, and analyzed frost lodging, melting, and other phenomena that appeared during the frost-formation process. The results showed that the frosting process on an arched surface can be divided into ice-film formation, rapid growth of the frost layer, and stable growth of the frost layer. Meanwhile, the phenomena of frost-branch breakage, lodging, and melting were observed. The surface temperature had a large effect on the frost formation and thickness of the frost layer, e.g., the formation time of the ice film on a surface at -5°C was the longest (~135 s), the frost layer formed on a surface at -20°C was the thickest (~660 μm). When microscopic observation of the frosting process was accompanied by calculation of the frost-layer thickness, it could be seen that the appearance of the frost branches was affected by the different thermal conductivities of the frost layers, undulating surface of the ice film, and temperature difference between the layers. The changes in the frost branches and the soft surface of the frost layer also affected the growth of the frost layer. The findings of this study are expected to provide guidelines for optimization of conventional defrosting methods.

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<![CDATA[Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein]]> https://www.researchpad.co/article/5c1d5b43d5eed0c4846eb351

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

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<![CDATA[An efficient proteome-wide strategy for discovery and characterization of cellular nucleotide-protein interactions]]> https://www.researchpad.co/article/5c12cef8d5eed0c484913c3b

Metabolite-protein interactions define the output of metabolic pathways and regulate many cellular processes. Although diseases are often characterized by distortions in metabolic processes, efficient means to discover and study such interactions directly in cells have been lacking. A stringent implementation of proteome-wide Cellular Thermal Shift Assay (CETSA) was developed and applied to key cellular nucleotides, where previously experimentally confirmed protein-nucleotide interactions were well recaptured. Many predicted, but never experimentally confirmed, as well as novel protein-nucleotide interactions were discovered. Interactions included a range of different protein families where nucleotides serve as substrates, products, co-factors or regulators. In cells exposed to thymidine, a limiting precursor for DNA synthesis, both dose- and time-dependence of the intracellular binding events for sequentially generated thymidine metabolites were revealed. Interactions included known cancer targets in deoxyribonucleotide metabolism as well as novel interacting proteins. This stringent CETSA based strategy will be applicable for a wide range of metabolites and will therefore greatly facilitate the discovery and studies of interactions and specificities of the many metabolites in human cells that remain uncharacterized.

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<![CDATA[Development and validation of a method for human papillomavirus genotyping based on molecular beacon probes]]> https://www.researchpad.co/article/5c0993d0d5eed0c4842ad9d7

We describe a new assaying system for the detection and genotyping of human papillomavirus (HPV) based on linear-after-the-exponential-PCR(LATE-PCR) and melting curve analysis. The 23 most prevalent HPV strains (types 6, 11, 16, 18, 31, 33, 35, 39, 42, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 81, 82, and 83) are assayed in two sealed reaction tubes within 2 h. Good sensitivity and specificity was evaluated by testing cloned HPV DNA and clinical samples. The detection limit was 5–500 copies/reaction depending on the genotype. No cross-reactivity was observed with the other HPV types that are not covered by our method or pathogens tested which were commonly found in female genital tract. When compared with the HPV GenoArray Diagnostic kit, the results from 1104 clinical samples suggest good overall agreement between the two methods,(98.37%, 95% CI: 97.44%–98.97%) and the kappa value was 0.954. Overall, this new HPV genotyping assay system presents a simple, rapid, universally applicable, sensitive, and highly specific detection methodology that should be useful for HPV detection and genotyping, therefore, is potentially of great value in clinical application.

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<![CDATA[Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues]]> https://www.researchpad.co/article/5989da0cab0ee8fa60b781b4

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.

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<![CDATA[A Real-Time PCR Method to Detect the Population Level of Halovirus SNJ1]]> https://www.researchpad.co/article/5989db1fab0ee8fa60bcedcf

Although viruses of haloarchaea are the predominant predator in hypersaline ecosystem, the culture studies about halovirus-host systems are infancy. The main reason is the tradition methodology (plaque assay) for virus-host interaction depends on culturable and susceptible host. Actually, more than 90% of haloarchaea are unculturable. Therefore, it is necessary to establish an approach for detecting the dynamics of virus in hypersaline environment without culture. In this study, we report a convenient method to determine the dynamics of halovirus SNJ1 based on quantitative real-time PCR (qPCR). All findings showed that the qPCR method was specific (single peak in melt curves), accurate (a good linear relationship between the log of the PFU and the Ct values, R2 = 0.99), reproducible (low coefficient of variations, below 1%). Additionally, the physicochemical characteristics of the samples tested did not influence the stability of qPCR. Therefore, the qPCR method has the potential value in quantifying and surveying haloviruses in halophilic ecological system.

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<![CDATA[Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents]]> https://www.researchpad.co/article/5989d9ecab0ee8fa60b6cee3

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

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<![CDATA[Association of HIV diversity and virologic outcomes in early antiretroviral treatment: HPTN 052]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf613

Higher HIV diversity has been associated with virologic outcomes in children on antiretroviral treatment (ART). We examined the association of HIV diversity with virologic outcomes in adults from the HPTN 052 trial who initiated ART at CD4 cell counts of 350–550 cells/mm3. A high resolution melting (HRM) assay was used to analyze baseline (pre-treatment) HIV diversity in six regions in the HIV genome (two in gag, one in pol, and three in env) from 95 participants who failed ART. We analyzed the association of HIV diversity in each genomic region with baseline (pre-treatment) factors and three clinical outcomes: time to virologic suppression after ART initiation, time to ART failure, and emergence of HIV drug resistance at ART failure. After correcting for multiple comparisons, we did not find any association of baseline HIV diversity with demographic, laboratory, or clinical characteristics. For the 18 analyses performed for clinical outcomes evaluated, there was only one significant association: higher baseline HIV diversity in one of the three HIV env regions was associated with longer time to ART failure (p = 0.008). The HRM diversity assay may be useful in future studies exploring the relationship between HIV diversity and clinical outcomes in individuals with HIV infection.

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<![CDATA[Thermodynamic Features of Structural Motifs Formed by β-L-RNA]]> https://www.researchpad.co/article/5989dab0ab0ee8fa60baaff4

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by 1H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.

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<![CDATA[Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling]]> https://www.researchpad.co/article/5989db17ab0ee8fa60bcd4ea

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

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<![CDATA[Superconductivity in Bismuth. A New Look at an Old Problem]]> https://www.researchpad.co/article/5989db4cab0ee8fa60bdab4e

To investigate the relationship between atomic topology, vibrational and electronic properties and superconductivity of bismuth, a 216-atom amorphous structure (a-Bi216) was computer-generated using our undermelt-quench approach. Its pair distribution function compares well with experiment. The calculated electronic and vibrational densities of states (eDOS and vDOS, respectively) show that the amorphous eDOS is about 4 times the crystalline at the Fermi energy, whereas for the vDOS the energy range of the amorphous is roughly the same as the crystalline but the shapes are quite different. A simple BCS estimate of the possible crystalline superconducting transition temperature gives an upper limit of 1.3 mK. The e-ph coupling is more preponderant in a-Bi than in crystalline bismuth (x-Bi) as indicated by the λ obtained via McMillan’s formula, λc = 0.24 and experiment λa = 2.46. Therefore with respect to x-Bi, superconductivity in a-Bi is enhanced by the higher values of λ and of eDOS at the Fermi energy.

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<![CDATA[The Cu-Li-Sn Phase Diagram: Isopleths, Liquidus Projection and Reaction Scheme]]> https://www.researchpad.co/article/5989db44ab0ee8fa60bd7ee5

The Cu-Li-Sn phase diagram was constructed based on XRD and DTA data of 60 different alloy compositions. Eight ternary phases and 14 binary solid phases form 44 invariant ternary reactions, which are illustrated by a Scheil-Schulz reaction scheme and a liquidus projection. Phase equilibria as a function of concentration and temperature are shown along nine isopleths. This report together with an earlier publication of our group provides for the first time comprehensive investigations of phase equilibria and respective phase diagrams. Most of the phase equilibria could be established based on our experimental results. Only in the Li-rich part where many binary and ternary compounds are present estimations had to be done which are all indicated by dashed lines. A stable ternary miscibility gap could be found which was predicted by modelling the liquid ternary phase in a recent work. The phase diagrams are a crucial input for material databases and thermodynamic optimizations regarding new anode materials for high-power Li-ion batteries.

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<![CDATA[Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood]]> https://www.researchpad.co/article/5989d9f2ab0ee8fa60b6ed15

Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer’s disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.

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<![CDATA[High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species]]> https://www.researchpad.co/article/5989db49ab0ee8fa60bd9adc

Background

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.

Methods/Principal Findings

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.

Conclusions/Significance

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.

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<![CDATA[Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease]]> https://www.researchpad.co/article/5989da45ab0ee8fa60b8b5b7

Background

High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.

Methods

A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression.

Results

All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.

Conclusion

MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.

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