scholarly journals Mlh1 haploinsufficiency induces microsatellite instability specifically in intestine

2019 ◽  
Author(s):  
Kul S. Shrestha ◽  
Elli-Mari Aska ◽  
Minna M. Tuominen ◽  
Liisa Kauppi
Keyword(s):  
Microsatellite Instability ◽  
Single Molecule ◽  
Age Of Onset ◽  
Complete Loss ◽  
Mouse Genetics ◽  
Dna Mismatch ◽  
Wide Range ◽  
Microsatellite Stability ◽  
Mlh1 Protein

AbstractTumors of Lynch syndrome (LS) patients display high levels of microsatellite instability (MSI), which results from complete loss of DNA mismatch repair (MMR), in line with Knudson’s two-hit hypothesis. Why some organs, in particular those of the gastrointestinal (GI) tract, are especially prone to tumorigenesis in LS remains unknown. We hypothesized that MMR is haploinsufficient in certain tissues, compromising microsatellite stability in a tissue-specific manner before tumorigenesis. Using mouse genetics, we tested how levels of MLH1, a central MMR protein, affect microsatellite stability in vivo and whether elevated MSI is detectable prior to loss of MMR function and to neoplastic growth. We assayed MSI by sensitive single-molecule PCR in normal jejunum and spleen of 4- and 12-month old Mlh1+/+, Mlh1+/− and Mlh1−/− mice, accompanied by measurements of Mlh1 mRNA and MLH1 protein expression levels.While spleen MLH1 levels of Mlh1+/− mice were, as expected, approximately 50% compared to wildtype mice, MLH1 levels in jejunum varied substantially between individual Mlh1+/− mice and decreased with age. Apparently, Mlh1+/− mice with soma-wide Mlh1 promoter methylation were the most venerable to MLH1 expression level decrease in jejunum. MLH1 levels (prior to complete loss of the protein) inversely correlated with MSI severity in Mlh1+/− jejunum, while in spleens of the same mice, MLH1 levels and microsatellites remained stable. Thus, Mlh1 haploinsufficiency affects specifically the intestine where MMR levels are particularly labile, inducing MSI in normal cells long before neoplasia. A similar mechanism likely also operates in the human GI epithelium, and could explain the wide range in age of onset of LS-associated tumorigenesis.

scholarly journals Prognostic significance of macrosatellite instability in patients under 50 years of age suffering from colorectal cancer

2009 ◽  
Vol 62 (5-6) ◽  
pp. 217-223
Author(s):  
Atila Fenjvesi
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Prognostic Significance ◽  
Young Patients ◽  
Dna Mismatch ◽  
Significant Difference ◽  
Sporadic Cases ◽  
Microsatellite Stability ◽  
Better Than

Introduction Colorectal cancer (CRC) can arise through two distinct mutational pathways: microsatellite instability or chromosomal instability. High-frequency microsatellite instability (MSI) occurs in approximately 15 percent of sporadic cases of CRCs. Many studies have well established that MSI, the hallmark of defective DNA mismatch repair, is associated with prolonged survival of CRC patients compared with tumors that are microsatellite stable. CRCs in patients under 50 years of age are rare and represent about 5% of the total number of tumors. The aim of this study was to analyze the prognostic significance of MSI in CRC patients younger than 50 at the time of diagnosis. Material and methods 31 patients with CRC under 50 years of age were tested for the presence of MSI, and compared with 35 patients aged 50 or more at the time of diagnosis. CRC-specific survival five-year- follow-up period was analyzed in relation to MSI status. Results The frequency of MSI among the young patients was 35.48%, which was significantly higher than the rate of 11.43% noted in older patients with CRCs (p<0.042). This study revealed no difference in survival in patients with CRCs aged less than 50 compared with those over 50 years of age. The five-years survival of young CRCs patients with MSI 81.82%, was better than that of the patients with cancers with microsatellite stability, 60%, but there was no significant difference in statistics. Discussion and conclusion In our study there was no statistically detectable significant difference between tumor microsatellite status and survival in young patients, although we confirmed the previous observations that MSI is associated with better prognosis. We found that the pathological stage of CRC was an independent and powerful predictor of the clinical outcome.

scholarly journals Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

2018 ◽  
Author(s):  
Achim Löf ◽  
Philipp U Walker ◽  
Steffen M Sedlak ◽  
Tobias Obser ◽  
Maria A Brehm ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Protein Domain ◽  
High Yield ◽  
Primary Hemostasis ◽  
Wide Range ◽  
And Function ◽  
Von Willebrand

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

scholarly journals FLEXIBILITY AND CONTROL OF PROTEIN–DNA LOOPS

2006 ◽  
Vol 01 (04) ◽  
pp. 327-341 ◽  
Author(s):  
JASON D. KAHN ◽  
RAYMOND CHEONG ◽  
RUCHI A. MEHTA ◽  
LAURENCE M. EDELMAN ◽  
MICHAEL A. MORGAN
Keyword(s):  
Single Molecule ◽  
Lac Repressor ◽  
Ring Closure ◽  
Dna Loops ◽  
Mobility Shift ◽  
Strong Constraint ◽  
Dna Loop ◽  
Wide Range ◽  
Dna Cyclization

Protein–DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)–DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI–DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI–DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than that expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI–DNA structure is strongly influenced by flanking DNA.

A Close-To-Native System to Study Membrane Protein Dynamics by Single-Molecule Optical Microscopy: an Application to Bacteriorhodopsin

2001 ◽  
Vol 7 (S2) ◽  
pp. 30-31
Author(s):  
Nicoletta Kahya ◽  
Eve I. Pécheur ◽  
Douwe A. Wiersma ◽  
Dick Hoekstra
Keyword(s):  
Optical Microscopy ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Transmembrane Protein ◽  
Passive Component ◽  
Signal Transducers ◽  
Wide Range ◽  
Minimum Number

Biological membranes are not just a passive component of the cells, they actively support their functioning as the imbedded protein machineries carry out a wide range of crucial biochemical processes. Although studies in vivo are becoming more and more accessible to single-molecule optical microscopy, in vitro studies are still very much informative for the understanding of individual biological machineries. One of the major goals is to define the minimum number of components of a machinery that is necessary for a particular step, thereby allowing detailed studies of the mechanism of action. Reconstitution of a transmembrane protein system in artificial membranes (liposomes) is the main method for such a strategy, which thus may provide the option to investigate the functioning of transport proteins, ion channels, fusion machineries, and signal transducers in relation to their environment.We present a novel procedure in order to reconstitute transmembrane proteins in chemically well-defined and close-to-native lipid bilayers, providing an in vitro system for single-molecule optical microscopy. Furthermore, an application of this technique is shown in the case of a single-molecule study of protein-protein and protein-lipid interactions for the light-induced proton pump bacteriorhodopsin.In this study, Giant Unilamellar Vesicles (GUV), 10-100 μm sized, are used as lipid bilayer models for several reasons.

scholarly journals Supercoiling DNA optically

2019 ◽  
Vol 116 (52) ◽  
pp. 26534-26539
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Supercoiling ◽  
Biological Interactions ◽  
Mitochondrial Transcription ◽  
Supercoiled Dna ◽  
Single Molecule Level ◽  
Mitochondrial Transcription Factor ◽  
Wide Range

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

TUMOUR PATHOLOGY PREDICTS MICROSATELLITE INSTABILITY IN A POPULATION-BASED SERIES OF COLORECTAL CANCER CASES

2008 ◽  
Vol 31 (4) ◽  
pp. 12
Author(s):  
A J Hyde ◽  
D Fontaine ◽  
R C Green ◽  
M Simms ◽  
P S Parfrey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Lynch Syndrome ◽  
Microsatellite Instability ◽  
Population Based ◽  
Pathological Features ◽  
Predictive Tool ◽  
Entire Cohort ◽  
Dna Mismatch ◽  
Bethesda Guidelines ◽  
Revised Bethesda Guidelines

Background: Lynch Syndrome is an autosomal dominant trait that accounts forapproximately 3% of all cases of colorectal cancer (CRC). It is caused by mutations in DNA mismatch repair (MMR) genes, most commonly MLH1 or MSH2. These MMR defects cause high levels of microsatellite instability (MSI-H) in the tumours. MSI testing of all CRCs to identify potential Lynch Syndrome cases is not practical, so the Bethesda Guidelines, which use clinical and pathological features, were created to identify those tumours most likely to be MSI-H^1. In 2007 Jenkins et. al. created MsPath, a tool based on the pathological features described in the rarely used 3^rd Bethesda criterion, to improve prediction of MSI-H tumours among CRC cases diagnosed before age 60 years^2. Methods: We collected a population-based cohort of 716 CRC cases diagnosed before age 75 years in Newfoundland. For each of these cases we collected family history, performed MSI analysis, and scored a number of pathological features for the purpose of evaluating the accuracy of the Bethesda Criteria and MsPath at predicting MSI-H tumours. Results: Our work validates the MsPath tool in the Newfoundland population for the same age group used to create the tool. We found it identified MSI-H cases with a sensitivity of 95% and specificity of 35% in our population of CRCcases diagnosed before age 60 years (n=290). We also tested this tool on our older population of CRCcases, diagnosed at ages 60 to 74 years (n=426). We found it to be at least as predictive in this population,with a sensitivity of 95% and a specificity of 42%. We then used our entire cohort (N=716) to compare MsPath with the other Bethesda criteria.Bethesda criteria 1, 2, 4 and 5 together predicted MSI-H cases with a sensitivity of 67% and a specificity of 51%. MsPath was better at identifying these cases, with a sensitivity of 95% and a specificity of 39%. Conclusions: We conclude that MsPath can be extended to include patients diagnosed with CRC before age 75 years. As well, we have found that MsPath is a better predictive tool than the Revised Bethesda Guidelines for identifying MSI-H cases within a population-based setting of colorectal cancer. References: 1. Umar, A. et. al. J Natl Cancer Inst 2004;96:261-8 2.Jenkins, M.A. et. al. Gastroenterology 2007;133:48-56

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

2018 ◽  
Author(s):  
Madushani Dharmarwardana ◽  
André F. Martins ◽  
Zhuo Chen ◽  
Philip M. Palacios ◽  
Chance M. Nowak ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Single Molecule ◽  
Biological Fluids ◽  
Cellular Respiration ◽  
Organic Radical ◽  
Paramagnetic Resonance ◽  
Metal Free ◽  
Disease States

Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by “click conjugating” paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced <i>in vivo</i> to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species—in particular superoxide—and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for <i>T<sub>1</sub></i> contrast at low field (<3.0 T), and <i>T<sub>2</sub></i> contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for “quenchless fluorescent” bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. <a>Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.</a>

Supramolecular Enhancement of Aminoxyl ORCA Contrast Agents

2019 ◽  
Author(s):  
Hamilton Lee ◽  
Jenica Lumata ◽  
Michael A. Luzuriaga ◽  
Candace Benjamin ◽  
Olivia Brohlin ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Side Effects ◽  
Magnetic Resonance ◽  
Tobacco Mosaic Virus ◽  
Contrast Agents ◽  
Single Molecule ◽  
Organic Radical ◽  
Resonance Imaging ◽  
Physiological Conditions

<div><div><div><p>Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further over came the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.</p></div></div></div>

Ethnomedicinal uses, Phytochemistry and Pharmacological Aspects of the Genus Berberis Linn: A Comprehensive Review

2020 ◽  
Vol 23 ◽  
Author(s):  
Roohi Mohi-ud-din ◽  
Reyaz Hassan Mir ◽  
Prince Ahad Mir ◽  
Saeema Farooq ◽  
Syed Naiem Raza ◽  
...  
Keyword(s):  
Chemical Constituents ◽  
Pharmacological Activities ◽  
Traditional Use ◽  
Comprehensive Review ◽  
Wide Range ◽  
Anti Cancer ◽  
Comprehensive Survey ◽  
Ethnomedicinal Uses

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.

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