N‐terminus of hMLH1 confers interaction of hMutLα and hMutLβ with hMutSα

Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSα (hMSH2‐hMSH6) and hMutLα (hMLH1‐hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSα recruits hMutLα, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLα as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSα using a novel interaction assay. We show that, although the interaction requires ATP, hMutLα does not need to hydrolyse this nucleotide to join hMutSα on DNA, suggesting that ATP hydrolysis by hMutLα happens downstream of complex formation. The analysis of the individual subunits of hMutLα demonstrated that the hMutSα–hMutLα interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N‐terminus of hMLH1 confers this interaction. In contrast, only the C‐terminus stabilised and co‐immunoprecipitated hPMS2 when both proteins were co‐expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C‐terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLβ (hMLH1–hPMS1). We show that hMutLβ interacts as efficiently with hMutSα as hMutLα, and that it predominantly binds to hMutSα via hMLH1 as well

Heat and Moisture Relevant In Situ Measurements in a Railway Passenger Vehicle Driving through the Swiss Alpine Region

Transportation is a major sector of energy consumption in most, if not in all, European countries. Besides the energy used for traction, energy is also consumed for ventilation, heating, and cooling inside the vehicles to assure traveler comfort. This issue gains increasing importance as the demand for public transport increases in the future. There is a need for retrofit to improve the thermal resistance of the envelope of existing vehicles to reduce the heat loss to the environment during the cold period of the year, especially in the Alpine region. A major concern in adding insulation material to the envelope is the possibility of convective moisture transfer due to air circulation in the vehicle, which would cause condensation accumulation on the cold surfaces. The present investigation addresses this topic by measuring surface and air temperature, air moisture, air flow, and heat flow at several critical locations of a vehicle during its travel in the Swiss Alpine region over several months during the cold period of the year. Temperature measurements showed the potential of reducing the heat losses in some parts of the vehicle. The level and duration of the moisture exposure did not suggest a relevant formation of condensation in the cross-section of the vehicle wall. The observed increase in relative humidity when driving through tunnels is too short to cause relevant condensation in the vehicle shell. The measured low air flow justifies the assumption that no forced convection occurs in the envelope cavities

Determinants of soluble angiotensin-converting enzyme 2 concentrations in adult patients with complex congenital heart disease

Background Angiotensin-converting enzyme (ACE) 2 is known to be a functional receptor for SARS-CoV-2 in the current pandemic. Soluble ACE2 (sACE2) concentrations are elevated in patients with various cardiovascular disorders including heart failure. Methods In a total of 182 consecutive adult patients with complex congenital heart disease (CHD) and 63 healthy controls, sACE2 concentrations were measured in serum using the Human ACE2® assay by Cloud-Clone Corporation and associated with clinical, laboratory and echocardiographic parameters. Results Median sACE2 levels were increased in patients with complex CHD as compared to healthy controls (761.9 pg/ml vs 365.2 pg/ml, p < 0.001). Moreover, sACE2 concentrations were significantly elevated in patients with a higher NYHA class ≥ III (1856.2 pg/ml vs 714.5 pg/ml in patients with NYHA class I/II, p < 0.001). Using linear regression analysis, higher sACE2 levels were associated with a higher NYHA class ≥ III, more severe CHD, a morphological left systemic ventricle, higher creatinine and the use of mineralocorticoid receptor antagonists (MRA) in the univariable model. The use of ACE inhibitors or angiotensin receptor blockers (ARB) was associated with lower sACE2 levels. In the multivariable model, higher sACE2 levels were independently associated with a higher NYHA class ≥ III (p = 0.002) and lower sACE2 levels with the use of ACE inhibitors or ARB (p = 0.001). Conclusion Soluble ACE2 concentrations were significantly increased in all types of complex CHD with highest levels found in patients with NYHA class ≥ III. Moreover, a higher NYHA class ≥ III was the most significant determinant that was independently associated with elevated sACE2 concentrations

MicroRNA-29b/c-3p Indicate Advanced Liver Fibrosis/Cirrhosis in Univentricular Heart Patients With and Without Fontan Palliation

Aim: The present study aims to identify those microRNAs (miRNAs) in patients with univentricular heart (UVH) disease with and without Fontan palliation that may be associated with advanced liver fibrosis/cirrhosis. Materials and Methods: SurePrintTM 8 × 60K Human v21 miRNA arrays were used to determine the miRNA abundance profiles in the blood of 48 UVH patients with and without Fontan palliation and 32 matched healthy controls. The abundance levels of selected miRNAs have been validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Results: According to microarray analysis, 50 miRNAs were found to be significantly abundant in UVH patients of which miR-29b-3p and miR-29c-3p were significantly related to the model of end-stage liver disease (MELD)-Albumin and albumin-bilirubin (ALBI) score representing advanced liver fibrosis/cirrhosis. Relative expression levels of both miRNAs were significantly higher in patients with a higher collapsibility index representing venous hepatic congestion, a higher MELD-Albumin or ALBI score and incomplete or no Fontan palliation. In the logistic regression analysis, a MELD-Albumin score ≥ 11 or ALBI score > −2.6 were best predicted by total bilirubin (OR 6.630, P = 0.016), albumin (OR 0.424, P = 0.026), and miR-29c-3p (OR 33.060, P = 0.047). After adjustment to the status of Fontan palliation, however, no statistical significance of these parameters was found thus underlining the importance of palliation status on progression of liver fibrosis/ cirrhosis in UVH patients. Conclusions: In UVH patients with and without Fontan palliation, miR-29b-3p and miR-29c-3p seem to be markers of advanced liver fibrosis/cirrhosis and thus may be used in the risk assessment of these patients

High Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides

On the potential of augmented reality for mathematics teaching with the application cleARmaths

Learning content in mathematics, such as vector geometry, is still predominantly taught in an abstract manner, as the visualization and interaction of three-dimensional problems are limited with classical forms of teaching such as blackboard lessons or exercise sheets. This research article proposes the use of augmented reality (AR) in mathematics education. The proposed approach aims at easing the learning process related to vector geometry currently taught in senior mathematics classes by using intuitive visualization. The article introduces the concept of AR and presents the didactic foundations and the influence on the learning process based on an extensive literature review. Although studies see great potential in the use of AR for teaching mathematics, the method has so far hardly been used in schools. This can be mainly explained by the technological entry barrier of AR and the lack of simple, robust AR applications, in particular for vector geometry. To fill this gap, the authors developed “cleARmaths”, a developed android application for augmented reality-based teaching in vector geometry that allows widespread use. As a didactical concept, some example exercises sessions with the app are proposed, demonstrating how the app could be used in a mathematics classroom. Finally, the app was evaluated in a mathematics class and the results analyzed in a detailed study. It was found by the teacher and students to be beneficial and amusing, demonstrating the potential for AR in mathematics classes

Strategies for plasma proteomic profiling of cancers

Despite a voluminous literature on potential protein biomarkers and a compelling need for diagnostic tests based on biomarkers to detect cancers at much earlier, more treatable stages, progress has been limited. New methods and new instruments for analysis of differences in gene expression, gene methylation, and proteomics are being employed to try to accelerate the discovery phase. Given the heterogeneity of tumor mechanisms and the limitations of analytical methods, it is likely that a variety of strategies will be needed and will be complementary. That is the basis of this review of proteomic approaches. This article adopts a systems biology view, starting with mRNA transcripts in tumors and cultured tumor cells to detect mRNA overexpression, some of which will be correlated with protein overexpression. Some of those proteins may be secreted or released into proximal biofluids and plasma. Detection of low-abundance tumor proteins in the complex and dynamic mixture that is plasma requires combinations of increasingly powerful technologies. The biological amplification of protein signals through the immune system offers autoantibodies as potential biomarkers. Higher abundance proteins, including acute-phase reactants, may have practical value, especially if the proteins are modified as part of the cancer processes. Low molecular weight proteins, fragments, and peptides may offer complementary biomarkers. Promising biomarker candidates must be confirmed in independent studies. Then they must be submitted to higher-throughput methods practical for large-scale validation studies and, hopefully, for clinical and epidemiological applications. Standardized operating procedures for specimen handling, design and use of various reference standards, care to avoid bias and confounding, and guidelines for reporting findings and contributing datasets should enhance the prospects for predictive proteomic profiling of people at risk for cancers.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55851/1/5662_ftp.pd

Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma

p53 alterations are considered to be predictive of poor prognosis in hepatocellular carcinoma (HCC) and may induce a humoral response. Anti-p53 serum antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) using purified recombinant human p53 on 130 European HCC patients before treatment and during the clinical course of the disease. p53 immunohistochemistry was performed on tumours from the 52 patients who underwent surgery, and DNA sequencing analysis was initiated when circulating anti-p53 antibodies were detected. Nine (7%) HCC patients had anti-p53 serum antibodies before treatment. During a mean period of 30 months of follow-up, all the negative patients remained negative, even when recurrence was observed. Of the nine positive patients, eight were still positive 12–30 months after surgery. The presence of anti-p53 serum antibodies was correlated neither with mutation of the p53 gene nor the serum alpha-fetoprotein levels and clinicopathological characterics of the tumours. However, a greater incidence of vascular invasion and accumulation of p53 protein were observed in the tumours of these patients (P < 0.03 and P < 0.01 respectively) as well as a better survival rate without recurrence (P = 0.05). In conclusion, as was recently shown in pancreatic cancer, anti-p53 serum antibodies may constitute a marker of relative ‘good prognosis’ in a subgroup of patients exhibiting one or several markers traditionally thought to be of bad prognosis. © 1999 Cancer Research Campaig