Pull-off characteristics of double-shanked compared to single-shanked ligation clips: an animal study.
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ABSTRACT:
SUBMITTER: Schenk M
PROVIDER: S-EPMC6753983 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:AimsThe coronary artery ligation model in rodents mimics human myocardial infarction (MI). Normally mechanical ventilation and prolonged anesthesia period are needed. Recently, a method has been developed to create MI by popping-out the heart (without ventilation) followed by immediate suture ligation. Mortality is high due to the time-consuming suture ligation process while the heart is exposed. We sought to improve this method and reduce mortality by rapid coronary ligation using a surgical clip instead of a suture.Methods and resultsMice were randomized into 3 groups: clip MI (CMI), suture MI (SMI), or sham (SHAM). In all groups, heart was manually exposed without intubation through a small incision on the chest wall. Unlike the conventional SMI method, mice in the CMI group received a metal clip on left anterior descending artery (LAD), quickly dispensed by an AutoSuture Surgiclip™. The CMI method took only 1/3 of ligation time of the standard SMI method and improved post-MI survival rate. TTC staining and Masson's trichrome staining revealed a similar degree of infarct size in the SMI and CMI groups. Echocardiograph confirmed that both SMI and CMI groups had a similar reduction of ejection fraction and fraction shortening over the time. Histological analysis showed that the numbers of CD68+ macrophages and apoptotic cells (TUNEL-positive) are indistinguishable between the two groups.ConclusionThis new method, taking only less than 3 minutes to complete, represents an efficient myocardial infarction model in rodents.
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Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes. 5 samples + 3 input samples
2010-11-01 | E-GEOD-18481 | biostudies-arrayexpress
Project description:Recent work has shown that cyanide ligation increases the redox potentials of Fe(4)S(4) clusters, enabling the isolation of [Fe(4)S(4)(CN)4]4-, the first synthetic Fe(4)S(4) cluster obtained in the all-ferrous oxidation state (Scott, T. A.; Berlinguette, C. P.; Holm, R. H.; Zhou, H.-C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9741). The generality of reduced cluster stabilization has been examined with MoFe(3)S(4) clusters. Reaction of single-cubane [(Tp)MoFe(3)S(4)(PEt(3))3]1+ and edge-bridged double-cubane [(Tp)2Mo(2)Fe(6)S(8)(PEt(3))4] with cyanide in acetonitrile affords [(Tp)MoFe(3)S(4)(CN)3]2- (2) and [(Tp)2Mo(2)Fe(6)S(8)(CN)4]4- (5), respectively. Reduction of 2 with KC(14)H(10) yields [(Tp)MoFe(3)S(4)(CN)3]3- (3). Clusters were isolated in approximately 70-90% yields as Et(4)N+ or Bu(4)N+ salts; clusters 3 and 5 contain all-ferrous cores, and 3 is the first [MoFe(3)S(4)]1+ cluster isolated in substance. The structures of 2 and 3 are very similar; the volume of the reduced cluster core is slightly larger (2.5%), a usual effect upon reduction of cubane-type Fe(4)S(4) and MFe(3)S(4) clusters. Redox potentials and 57Fe isomer shifts of [(Tp)MoFe(3)S(4)L3]2-,3- and [(Tp)2Mo(2)Fe(6)S(8)L(4)]4-,3- clusters with L = CN-, PhS-, halide, and PEt3 are compared. Clusters with pi-donor ligands (L = halide, PhS) exhibit larger isomer shifts and lower (more negative) redox potentials, while pi-acceptor ligands (L = CN, PEt3) induce smaller isomer shifts and higher (less-negative) redox potentials. When the potentials of 3/2 and [(Tp)MoFe(3)S(4)(SPh)3]3-/2- are compared, cyanide stabilizes 3 by 270 mV versus the reduced thiolate cluster, commensurate with the 310 mV stabilization of [Fe(4)S(4)(CN)4]4- versus [Fe(4)S(4)(SPh)4]4- where four ligands differ. These results demonstrate the efficacy of cyanide stabilization of lower cluster oxidation states. (Tp = hydrotris(pyrazolyl)borate(1-)).
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Project description:This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.
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Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes.
2010-11-01 | GSE18481 | GEO
Project description:Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.
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Project description:BackgroundAlthough single and double lung transplantation outcomes for chronic obstructive pulmonary disease (COPD) have been investigated, right and left single lung transplants have never been rigorously compared to evaluate disease-specific differences. Single lung transplants for COPD often have hyperinflation of the contralateral native lung, which may be more pronounced in left lung transplants.MethodsUsing the United Network for Organ Sharing registry, we conducted a retrospective cohort study of 5,585 adults who underwent lung transplantation for COPD from May 4, 2005 to June 30, 2017. Subjects were followed until March 2019. Post-transplant survival was compared using Cox proportional hazards and Royston and Parmar's flexible parametric survival models. We adjusted for donor and recipient factors with known or plausible associations with survival.ResultsLung transplant recipients who received a left single lung transplant for COPD had an increased risk of post-transplant death when compared with those who received a right single lung transplant for COPD (hazard ratio [HR]: 1.24, 95% CI: 1.08-1.48, p = 0.002). Survival did not differ significantly between double lung transplant and right single lung transplant recipients (HR: 0.88, 95% CI: 0.77-1.02, p = 0.086). Adjusted 5-year survival was 57.8% (95% CI: 55.7-60.1) for double lung recipients, 56.7% (95% CI: 55.4-58.0) for right single lung recipients, and 50.9% (95% CI: 47.2-55.0) for left single lung recipients.ConclusionsIn COPD, right single lung transplantation was associated with improved post-transplant survival compared with left single lung transplantation, and no significant difference in post-transplant survival compared with double lung transplantation was found. In light of the ongoing donor lung shortage, preferential allocation of right single lungs to patients with COPD should be considered.
| S-EPMC7863775 | biostudies-literature
Project description:BACKGROUND:Patients with colorectal cancer (CRC) with mismatch repair-deficient (dMMR) tumours without MLH1 methylation or germline MMR pathogenic variants (PV) were previously thought to have Lynch syndrome (LS). It is now appreciated that they can have double somatic (DS) MMR PVs. We explored the clinical characteristics between patients with DS tumours and LS in two population-based cohorts. METHODS:We included patients with CRC from Ohio 2013-2016 and Iceland 2000-2009. All had microsatellite instability testing and/or immunohistochemistry (IHC) of MMR proteins, and MLH1 methylation testing when indicated. Germline next-generation sequencing was performed for all with dMMR tumours; tumour sequencing followed for patients with unexplained dMMR. Clinical characteristics of DS patients and patients with LS were compared. RESULTS:Of the 232 and 51 patients with non-methylated dMMR tumours in the Ohio and Iceland cohorts, respectively, 57.8% (n=134) and 45.1% (n=23) had LS, 32.8% (n=76) and 31.4% (n=16) had DS PVs, 6% (n=14) and 9.8% (n=5) were unexplained and 4.3% (n=10) and 13.7% (n=7) had incorrect IHC. Age of diagnosis for DS patients was older than patients with LS (p=3.73×10-4) in the two cohorts. Patients with LS were more likely to meet Amsterdam II criteria (OR=15.81, p=8.47×10-6) and have multiple LS-associated tumours (OR=6.67, p=3.31×10-5). Absence of MLH1/PMS2 was predictive of DS PVs; isolated MSH6 and PMS2 absence was predictive of LS in both cohorts. CONCLUSIONS:Individuals with LS are 15× more likely to meet Amsterdam II criteria and >5×?more likely to have multiple cancers as compared with those with DS tumours. Furthermore, isolated loss of MSH6 or PMS2 protein predicts LS.
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Project description:OBJECTIVES:Prospective registration of animal studies has been suggested as a new measure to increase value and reduce waste in biomedical research. We sought to further explore and quantify animal researchers' attitudes and preferences regarding animal study registries (ASRs). DESIGN:Cross-sectional online survey. SETTING AND PARTICIPANTS:We conducted a survey with three different samples representing animal researchers: i) corresponding authors from journals with high Eigenfactor, ii) a random Pubmed sample and iii) members of the CAMARADES network. MAIN OUTCOME MEASURES:Perceived level of importance of different aspects of publication bias, the effect of ASRs on different aspects of research as well as the importance of different research types for being registered. RESULTS:The survey yielded responses from 413 animal researchers (response rate 7%). The respondents indicated, that some aspects of ASRs can increase administrative burden but could be outweighed by other aspects decreasing this burden. Animal researchers found it more important to register studies that involved animal species with higher levels of cognitive capabilities. The time frame for making registry entries publicly available revealed a strong heterogeneity among respondents, with the largest proportion voting for "access only after consent by the principal investigator" and the second largest proportion voting for "access immediately after registration". CONCLUSIONS:The fact that the more senior and experienced animal researchers participating in this survey clearly indicated the practical importance of publication bias and the importance of ASRs underscores the problem awareness across animal researchers and the willingness to actively engage in study registration if effective safeguards for the potential weaknesses of ASRs are put into place. To overcome the first-mover dilemma international consensus statements on how to deal with prospective registration of animal studies might be necessary for all relevant stakeholder groups including animal researchers, academic institutions, private companies, funders, regulatory agencies, and journals.
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