166 research outputs found
Multilevel Deconstruction of the In Vivo Behavior of Looped DNA-Protein Complexes
Protein-DNA complexes with loops play a fundamental role in a wide variety of
cellular processes, ranging from the regulation of DNA transcription to
telomere maintenance. As ubiquitous as they are, their precise in vivo
properties and their integration into the cellular function still remain
largely unexplored. Here, we present a multilevel approach that efficiently
connects in both directions molecular properties with cell physiology and use
it to characterize the molecular properties of the looped DNA-lac repressor
complex while functioning in vivo. The properties we uncover include the
presence of two representative conformations of the complex, the stabilization
of one conformation by DNA architectural proteins, and precise values of the
underlying twisting elastic constants and bending free energies. Incorporation
of all this molecular information into gene-regulation models reveals an
unprecedented versatility of looped DNA-protein complexes at shaping the
properties of gene expression.Comment: Open Access article available at
http://www.plosone.org/article/fetchArticle.action?articleURI=info%3Adoi%2F10.1371%2Fjournal.pone.000035
Towards a target label-free suboptimum oligonucleotide displacement-based detection system
A novel method for the future development of label-free DNA sensors is proposed here. The approach is based on the displacement of a labelled suboptimum mutated oligonucleotide hybridised with the immobilised biotin-capture probe. The target fully complementary to the biotin-capture probe can displace the labelled oligonucleotide causing a subsequent decrease of the signal that verifies the presence of the target. The decrease of signal was demonstrated to be proportional to the target concentration. A study of the hybridisation of mutated and complementary labelled oligonucleotides with an immobilised biotin-capture probe was carried out. Different kinetic and thermodynamic behaviour was observed for heterogeneous hybridisation of biotin-capture probe with complementary or suboptimum oligonucleotides. The displacement method evaluated colourimetrically achieved the objective of decreasing the response time from 1 h for direct hybridisation of 19-mer oligonucleotides in the direct enzyme-linked oligonucleotide assay (ELONA) to 5 min in the case of displacement detection in the micromolar concentration range
Spatial and topological organization of DNA chains induced by gene co-localization
Transcriptional activity has been shown to relate to the organization of
chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In
particular, highly transcribed genes, RNA polymerases and transcription factors
gather into discrete spatial foci called transcription factories. However, the
mechanisms underlying the formation of these foci and the resulting topological
order of the chromosome remain to be elucidated. Here we consider a
thermodynamic framework based on a worm-like chain model of chromosomes where
sparse designated sites along the DNA are able to interact whenever they are
spatially close-by. This is motivated by recurrent evidence that there exists
physical interactions between genes that operate together. Three important
results come out of this simple framework. First, the resulting formation of
transcription foci can be viewed as a micro-phase separation of the interacting
sites from the rest of the DNA. In this respect, a thermodynamic analysis
suggests transcription factors to be appropriate candidates for mediating the
physical interactions between genes. Next, numerical simulations of the polymer
reveal a rich variety of phases that are associated with different topological
orderings, each providing a way to increase the local concentrations of the
interacting sites. Finally, the numerical results show that both
one-dimensional clustering and periodic location of the binding sites along the
DNA, which have been observed in several organisms, make the spatial
co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on
http://dx.doi.org/10.1371/journal.pcbi.100067
Systemic mastocytosis associated with t(8;21)(q22;q22) acute myeloid leukemia
Although KIT mutations are present in 20–25% of cases of t(8;21)(q22;q22) acute myeloid leukemia (AML), concurrent development of systemic mastocytosis (SM) is exceedingly rare. We examined the clinicopathologic features of SM associated with t(8;21)(q22;q22) AML in ten patients (six from our institutions and four from published literature) with t(8;21) AML and SM. In the majority of these cases, a definitive diagnosis of SM was made after chemotherapy, when the mast cell infiltrates were prominent. Deletion 9q was an additional cytogenetic abnormality in four cases. Four of the ten patients failed to achieve remission after standard chemotherapy and seven of the ten patients have died of AML. In the two patients who achieved durable remission after allogeneic hematopoietic stem cell transplant, recipient-derived neoplastic bone marrow mast cells persisted despite leukemic remission. SM associated with t(8;21) AML carries a dismal prognosis; therefore, detection of concurrent SM at diagnosis of t(8;21) AML has important prognostic implications
Knowledge Management and Organisational Culture
This international Handbook provides a comprehensive overview of key topics, debates and issues within the now well-established field of Knowledge Management (KM)
Structural Organization of DNA in Chlorella Viruses
Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes
Intramolecular Folding in Human ILPR Fragment with Three C-Rich Repeats
Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures
Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO
The last three decades has seen some important
advances in our ability to represent the conformation of
proteins in solution on the basis of hydrodynamic measurements.
Advances in theoretical modeling capabilities have
been matched by commensurate advances in the precision of
hydrodynamic measurements. We consider the advances in
whole-body (simple ellipsoid-based) modeling—still useful
for providing an overall idea of molecular shape, particularly
for those systems where only a limited amount of data is
available—and outline the ELLIPS suite of algorithms
which facilitates the use of this approach. We then focus
on bead modeling strategies, particularly the surface or
shell–bead approaches and the HYDRO suite of algorithms.
We demonstrate how these are providing great insights into
complex issues such as the conformation of immunoglobulins
and other multi-domain complexes
Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior
α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain
Collaborative care for the detection and management of depression among adults with hypertension in South Africa: study protocol for the PRIME-SA randomised controlled trial
Background: The high co-morbidity of mental disorders, particularly depression, with non-communicable diseases (NCDs) such as cardiovascular disease (CVD), is concerning given the rising burden of NCDs globally, and the role depression plays in confounding prevention and treatment of NCDs. The objective of this randomised control trial (RCT) is to determine the real-world effectiveness of strengthened depression identification and management on depression outcomes in hypertensive patients attending primary health care (PHC) facilities in South Africa (SA). Methods/design: The study design is a pragmatic, two-arm, parallel-cluster RCT, the unit of randomisation being the clinics, with outcomes being measured for individual participants. The 20 largest eligible clinics from one district in the North West Province are enrolled in the trial. Equal numbers of hypertensive patients (n = 50) identified as having depression using the Patient Health Questionnaire (PHQ-9) are enrolled from each clinic, making up a total of 1000 participants with 500 in each arm. The nurse clinicians in the control facilities receive the standard training in Primary Care 101 (PC101), a clinical decision support tool for integrated chronic care that includes guidelines for hypertension and depression care. Referral pathways available include referrals to PHC physicians, clinical or counselling psychologists and outpatient psychiatric and psychological services. In the intervention clinics, this training is supplemented with strengthened training in the depression components of PC101 as well as training in clinical communication skills for nurse-led chronic care. Referral pathways are strengthened through the introduction of a facility-based behavioural health counsellor, trained to provide structured manualised counselling for depression and adherence counselling for all chronic conditions. The primary outcome is defined as at least 50% reduction in PHQ-9 score measured at 6 months. Discussion: This trial should provide evidence of the real world effectiveness of strengtheneddepression identification and collaborative management on health outcomes of hypertensive patients withcomorbid depression attending PHC facilities in South Africa
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