DNA binding properties of a chemically synthesized DNA binding domain of hRFX1

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (∼90 mg) of pure compound. Preliminary two-dimensional 1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5′-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (Ka = 4×105/M/s), a dissociation constant (Kd = 6×10−2/s) and an equilibrium dissociation constant (KD = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16°C, illustrating stabilization of the double-stranded conformation induced by the peptid

Scalable and flexible inference framework for stochastic dynamic single-cell models

Understanding the inherited nature of how biological processes dynamically change over time and exhibit intra- and inter-individual variability, due to the different responses to environmental stimuli and when interacting with other processes, has been a major focus of systems biology. The rise of single-cell fluorescent microscopy has enabled the study of those phenomena. The analysis of single-cell data with mechanistic models offers an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. However, extracting mechanistic information from single-cell data has proven difficult. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic (e.g. variations in chemical reactions) and extrinsic (e.g. variability in protein concentrations) noise. Although several inference methods exist, the availability of efficient, general and accessible methods that facilitate modelling of single-cell data, remains lacking. Here we present a scalable and flexible framework for Bayesian inference in state-space mixed-effects single-cell models with stochastic dynamic. Our approach infers model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. We demonstrate the relevance of our approach by studying how cell-to-cell variation in carbon source utilisation affects heterogeneity in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway. We identify hexokinase activity as a source of extrinsic noise and deduce that sugar availability dictates cell-to-cell variability

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associ- ated with Parkinson’s disease, chronic inflammation and mycobac- terial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobac- terial control independently of autophagy. In vivo, LRRK2 defi- ciency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2- dependent cellular pathway that controls M. tuberculosis replica- tion by regulating phagosome maturation

mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In this study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein Leucine Rich Repeat Kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity

Efficacy and safety of statin therapy in older people: a meta-analysis of individual participant data from 28 randomised controlled trials

Background: Statin therapy has been shown to reduce major vascular events and vascular mortality in a wide range of individuals, but there is uncertainty about its efficacy and safety among older people. We undertook a meta-analysis of data from all large statin trials to compare the effects of statin therapy at different ages. Methods: In this meta-analysis, randomised trials of statin therapy were eligible if they aimed to recruit at least 1000 participants with a scheduled treatment duration of at least 2 years. We analysed individual participant data from 22 trials (n=134 537) and detailed summary data from one trial (n=12 705) of statin therapy versus control, plus individual participant data from five trials of more intensive versus less intensive statin therapy (n=39 612). We subdivided participants into six age groups (55 years or younger, 56–60 years, 61–65 years, 66–70 years, 71–75 years, and older than 75 years). We estimated effects on major vascular events (ie, major coronary events, strokes, and coronary revascularisations), cause-specific mortality, and cancer incidence as the rate ratio (RR) per 1·0 mmol/L reduction in LDL cholesterol. We compared proportional risk reductions in different age subgroups by use of standard χ2 tests for heterogeneity when there were two groups, or trend when there were more than two groups. Findings: 14 483 (8%) of 186 854 participants in the 28 trials were older than 75 years at randomisation, and the median follow-up duration was 4·9 years. Overall, statin therapy or a more intensive statin regimen produced a 21% (RR 0·79, 95% CI 0·77–0·81) proportional reduction in major vascular events per 1·0 mmol/L reduction in LDL cholesterol. We observed a significant reduction in major vascular events in all age groups. Although proportional reductions in major vascular events diminished slightly with age, this trend was not statistically significant (ptrend=0·06). Overall, statin or more intensive therapy yielded a 24% (RR 0·76, 95% CI 0·73–0·79) proportional reduction in major coronary events per 1·0 mmol/L reduction in LDL cholesterol, and with increasing age, we observed a trend towards smaller proportional risk reductions in major coronary events (ptrend=0·009). We observed a 25% (RR 0·75, 95% CI 0·73–0·78) proportional reduction in the risk of coronary revascularisation procedures with statin therapy or a more intensive statin regimen per 1·0 mmol/L lower LDL cholesterol, which did not differ significantly across age groups (ptrend=0·6). Similarly, the proportional reductions in stroke of any type (RR 0·84, 95% CI 0·80–0·89) did not differ significantly across age groups (ptrend=0·7). After exclusion of four trials which enrolled only patients with heart failure or undergoing renal dialysis (among whom statin therapy has not been shown to be effective), the trend to smaller proportional risk reductions with increasing age persisted for major coronary events (ptrend=0·01), and remained non-significant for major vascular events (ptrend=0·3). The proportional reduction in major vascular events was similar, irrespective of age, among patients with pre-existing vascular disease (ptrend=0·2), but appeared smaller among older than among younger individuals not known to have vascular disease (ptrend=0·05). We found a 12% (RR 0·88, 95% CI 0·85–0·91) proportional reduction in vascular mortality per 1·0 mmol/L reduction in LDL cholesterol, with a trend towards smaller proportional reductions with older age (ptrend=0·004), but this trend did not persist after exclusion of the heart failure or dialysis trials (ptrend=0·2). Statin therapy had no effect at any age on non-vascular mortality, cancer death, or cancer incidence. Interpretation: Statin therapy produces significant reductions in major vascular events irrespective of age, but there is less direct evidence of benefit among patients older than 75 years who do not already have evidence of occlusive vascular disease. This limitation is now being addressed by further trials. Funding: Australian National Health and Medical Research Council, National Institute for Health Research Oxford Biomedical Research Centre, UK Medical Research Council, and British Heart Foundation

Array-based capture, distribution, counting and multiplexed assaying of beads on a centrifugal microfluidic platform

We present a novel centrifugal microfluidic platform for the highly efficient manipulation and analysis of particles for applications in bead-based assays. The platform uses an array of geometrical V-cup barriers to trap particles using stopped-flow sedimentation under highly reproducible hydrodynamic conditions. The impact parameters governing the occupancy distribution and capture efficiency of the arrayed traps are investigated. The unique, nearly 100% capture efficiency paired with the capability to establish sharply peaked, single occupancy distributions enables a novel, digital readout mode for color-multiplexed, particle-based assays with low-complexity instrumentation. The presented technology marks an essential step towards a versatile platform for the integration of bead- and cell-based biological assays

An integrative simulation concept for extrusion blow molded plastic bottles

It is well known that the mechanical properties of extrusion blow molded plastic bottles are influenced by the process conditions, which leads to local orthotropic process-dependent material parameters. In this study, these parameters as well as local orientation effects are for the first time considered in the structural analysis of extrusion blow molded products. Furthermore, an integrative simulation concept has been worked out which combines process simulation and structural analysis to take the process history of the plastic bottle into account. The elastic modulus is described as a function of process parameters like the local degrees of stretching and the mold temperature. Because the local degrees of stretching differ considerably, a new algorithm was developed which identif ies the local degrees of stretching as well as their orientation using the results of the process simulation. For the data mapping between the process mesh and the structural analysis mesh, the self-developed MpCCI Mapper software was used. In a simulation of a top load test, the integrative simulation model was compared with the conventional model as well as with experimental data. Although in this particular case the influence of the orthotropic material model with respect to the local degrees of stretching is rather low, the general use of process dependent material parameters leads to much better results

Complete nucleotide sequence of the chlorarachniophyte nucleomorph: Nature’s smallest nucleus

The introduction of plastids into different heterotrophic protists created lineages of algae that diversified explosively, proliferated in marine and freshwater environments, and radically altered the biosphere. The origins of these secondary plastids are usually inferred from the presence of additional plastid membranes. However, two examples provide unique snapshots of secondary-endosymbiosis-in-action, because they retain a vestige of the endosymbiont nucleus known as the nucleomorph. These are chlorarachniophytes and cryptomonads, which acquired their plastids from a green and red alga respectively. To allow comparisons between them, we have sequenced the nucleomorph genome from the chlorarachniophyte Bigelowiella natans: at a mere 373,000 bp and with only 331 genes, the smallest nuclear genome known and a model for extreme reduction. The genome is eukaryotic in nature, with three linear chromosomes containing densely packed genes with numerous overlaps. The genome is replete with 852 introns, but these are the smallest introns known, being only 18, 19, 20, or 21 nt in length. These pygmy introns are shown to be miniaturized versions of normal-sized introns present in the endosymbiont at the time of capture. Seventeen nucleomorph genes encode proteins that function in the plastid. The other nucleomorph genes are housekeeping entities, presumably underpinning maintenance and expression of these plastid proteins. Chlorarachniophyte plastids are thus serviced by three different genomes (plastid, nucleomorph, and host nucleus) requiring remarkable coordination and targeting. Although originating by two independent endosymbioses, chlorarachniophyte and cryptomonad nucleomorph genomes have converged upon remarkably similar architectures but differ in many molecular details that reflect two distinct trajectories to hypercompaction and reduction