Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy ...of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.
Genome browsers facilitate integrated analysis of multiple genomics datasets yet visualize only a few regions at a time and lack statistical functions for extracting meaningful information. We ...present HiCognition, a visual exploration and machine-learning tool based on a new genomic region set concept, enabling detection of patterns and associations between 3D chromosome conformation and collections of 1D genomics profiles of any type. By revealing how transcription and cohesion subunit isoforms contribute to chromosome conformation, we showcase how the flexible user interface and machine learning tools of HiCognition help to understand the relationship between the structure and function of the genome.
The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)
...analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells
, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
Abstract
Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming ...daughter cells
1–3
. Assembly of mitotic chromosomes involves DNA looping by condensin
4–8
and chromatin compaction by global histone deacetylation
9–13
. Although condensin confers mechanical resistance to spindle pulling forces
14–16
, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.
Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same ...nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule‐based spindle can move them to opposite poles. While the spindle‐mediated transport of sister chromatids has been studied in detail, the chromosome‐intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop‐extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis‐specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.
Synopsis
The two DNA copies contained in replicated chromosomes need to be disentangled and packaged into separate thread‐like sister chromatids such that the microtubule‐based spindle can segregate one genome copy to each daughter cell during mitosis. This work shows that sister chromatid resolution initiates already during interphase via a process that depends on cohesin's DNA loop extrusion activity.
Cohesin resolves sister chromatids during G2 phase.
Hyperactivation of cohesin's looping processivity shapes mitosis‐like sister chromatids in G2 phase, independently of condensin.
Sister‐chromatid‐resolved Hi‐C shows that cohesin can separate sister chromatids over several megabases.
These findings support a model of DNA loop extrusion as a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes.
Mitosis‐like sister chromatids are shaped already during interphase, independently of condensin.
Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A ...notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.
We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.
The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To investigate whether gravitational valves reduce the risk of overdrainage complications compared with programmable valves in ventriculoperitoneal (VP) shunt surgery for idiopathic normal pressure ...hydrocephalus (iNPH).
Patients with iNPH may benefit from VP shunting but are prone to overdrainage complications during posture changes. Gravitational valves with tantalum balls are considered to reduce the risk of overdrainage but their clinical effectiveness is unclear.
We conducted a pragmatic, randomised, multicentre trial comparing gravitational with non-gravitational programmable valves in patients with iNPH eligible for VP shunting. The primary endpoint was any clinical or radiological sign (headache, nausea, vomiting, subdural effusion or slit ventricle) of overdrainage 6 months after randomisation. We also assessed disease specific instruments (Black and Kiefer Scale) and Physical and Mental Component Scores of the Short Form 12 (SF-12) generic health questionnaire.
We enrolled 145 patients (mean (SD) age 71.9 (6.9) years), 137 of whom were available for endpoint analysis. After 6 months, 29 patients in the standard and five patients in the gravitational shunt group developed overdrainage (risk difference -36%, 95% CI -49% to -23%; p<0.001). This difference exceeded predetermined stopping rules and resulted in premature discontinuation of patient recruitment. Disease specific outcome scales did not differ between the groups although there was a significant advantage of the gravitational device in the SF-12 Mental Component Scores at the 6 and 12 month visits.
Implanting a gravitational rather than another type of valve will avoid one additional overdrainage complication in about every third patient undergoing VP shunting for iNPH.
Context: The contribution of insulin resistance per se to the vascular risk conferred by the metabolic syndrome (MetS) is not known; conversely, it is uncertain whether insulin resistance confers ...vascular risk beyond the entity of the MetS.
Objective: The objective of this study was to investigate the impact of the MetS (Adult Treatment Panel III criteria) and insulin resistance (as estimated by the homeostasis model assessment index) on the incidence of vascular events.
Design and Patients: This was a prospective cohort study enrolling 750 consecutive patients undergoing coronary angiography for the evaluation of coronary artery disease.
Setting: The study was performed at a tertiary care clinical research center.
Main Outcome Measure: The main outcome measure was the incidence of vascular events over 2.3 yr.
Results: Both the MetS and insulin resistance predicted vascular events after controlling for non-MetS risk factors hazard ratio (HR), 2.74 (95% confidence interval, 1.71–4.39; P < 0.001) and 1.51 (1.24–1.84; P < 0.001), respectively. After additional adjustment for insulin resistance, the MetS remained significantly predictive of vascular events HR, 2.69 (1.57–4.64); P < 0.001, and conversely, insulin resistance remained significantly predictive of vascular events despite adjustment for the MetS standardized HR, 1.41 (1.14–1.75); P = 0.002. Additional adjustment for the presence of type 2 diabetes revealed that both the MetS adjusted HR, 2.57 (1.47–4.51); P = 0.001 and homeostasis model assessment of insulin resistance standardized adjusted HR, 1.37 (1.09–1.73); P = 0.007 significantly predicted vascular events independent from diabetes status.
Conclusions: Both the MetS and insulin resistance are strong and mutually independent predictors of vascular risk among angiographed coronary patients.
Is Atherosclerosis in Diabetes and Impaired Fasting Glucose Driven by Elevated LDL Cholesterol or by Decreased HDL Cholesterol?
Heinz Drexel , MD 1 2 ,
Stefan Aczel , MD 1 2 ,
Thomas Marte , MD 1 ,
...Werner Benzer , MD 2 ,
Peter Langer , PHD 1 ,
Willi Moll , MD 3 and
Christoph H. Saely , MD 1 2
1 Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria
2 Department of Medicine, Academic Teaching Hospital Feldkirch, Feldkirch, Austria
3 Institute for Clinical Chemistry, Feldkirch, Austria
Address correspondence and reprint requests to Heinz Drexel, MD, Professor of Medicine, VIVIT, Carinagasse 47, A-6800 Feldkirch,
Austria. E-mail: vivit{at}lkhf.at
Abstract
OBJECTIVE —To evaluate the atherogenicity of lipids in coronary patients with normal fasting glucose (NFG), impaired fasting glucose
(IFG), and type 2 diabetes.
RESEARCH DESIGN AND METHODS —Serum lipid values, the presence of angiographic coronary artery disease (CAD) at baseline, and the incidence of vascular
events over 2.3 years were recorded in 750 consecutive patients undergoing coronary angiography.
RESULTS —Triglycerides significantly ( P < 0.001) increased and HDL cholesterol ( P < 0.001) as well as LDL particle diameter ( P < 0.001) significantly decreased from subjects with NFG <5.6 mmol/l ( n = 272) over patients with IFG ≥5.6 mmol/l ( n = 314) to patients with type 2 diabetes ( n = 164). Factor analysis revealed two factors in the lipid profiles of our patients: triglycerides, HDL cholesterol, apolipoprotein
A1, and LDL particle diameter loaded high on an HDL-related factor, and total cholesterol, LDL cholesterol, and apolipoprotein
B loaded high on an LDL-related factor. In patients with type 2 diabetes, the HDL-related factor (odds ratio 0.648 95% CI
0.464–0.904; P = 0.011), but not the LDL-related factor (0.921 0.677–1.251; P = 0.597), was associated with significant coronary stenoses ≥50%. Consistently, in the prospective study, the HDL-related
factor (0.708 0.506–0.990; P = 0.044), but not the LDL-related factor (1.362 0.985–1.883; P = 0.061), proved significantly predictive for vascular events in patients with type 2 diabetes.
CONCLUSIONS —The low HDL cholesterol/high triglyceride pattern is associated with the degree of hyperglycemia. In coronary patients with
type 2 diabetes, this pattern correlates with the prevalence of CAD and significantly predicts the incidence of vascular events.
CAD, coronary artery disease
IFG, impaired fasting glucose
NFG, normal fasting glucose
Footnotes
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.
Accepted October 5, 2004.
Received July 30, 2004.
DIABETES CARE
Interleukin-10 (IL-10) signaling genes are attractive inflammatory bowel disease (IBD) candidate genes as IL-10 restricts intestinal inflammation, IL-10 polymorphisms have been associated with IBD in ...genome-wide association studies, and mutations in IL-10 and IL-10 receptor (IL-10R) genes have been reported in immunodeficient children with severe infantile-onset IBD. Our objective was to determine if IL-10R polymorphisms were associated with early-onset IBD (EO-IBD) and very-early-onset IBD (VEO-IBD).
Candidate-gene analysis of IL10RA and IL10RB was performed after initial sequencing of an infantile onset-IBD patient identified a novel homozygous mutation. The discovery cohort included 188 EO-IBD subjects and 188 healthy subjects. Polymorphisms associated with IBD in the discovery cohort were genotyped in an independent validation cohort of 422 EO-IBD subjects and 480 healthy subjects.
We identified a homozygous, splice-site point mutation in IL10RA in an infantile-onset IBD patient causing a premature stop codon (P206X) and IL-10 insensitivity. IL10RA and IL10RB sequencing in the discovery cohort identified five IL10RA polymorphisms associated with ulcerative colitis (UC) and two IL10RB polymorphisms associated with Crohn's disease (CD). Of these polymorphisms, two IL10RA single nucleotide polymorphisms, rs2228054 and rs2228055, were associated with VEO-UC in the discovery cohort and replicated in an independent validation cohort (odds ratio OR 3.08, combined P = 2 x 10(-4); and OR 2.93, P = 6 x 10(-4), respectively).
We identified IL10RA polymorphisms that confer risk for developing VEO-UC. Additionally, we identified the first splice site mutation in IL10RA resulting in infantile-onset IBD. This study expands the phenotype of IL10RA polymorphisms to include both severe arthritis and VEO-UC.
PDF
