Estimating parameters of speciation models based on refined summaries of the joint site-frequency spectrum

Understanding the processes and conditions under which populations diverge to give rise to distinct species is a central question in evolutionary biology. Since recently diverged populations have high levels of shared polymorphisms, it is challenging to distinguish between recent divergence with no (or very low) inter-population gene flow and older splitting events with subsequent gene flow. Recently published methods to infer speciation parameters under the isolation-migration framework are based on summarizing polymorphism data at multiple loci in two species using the joint site-frequency spectrum (JSFS). We have developed two improvements of these methods based on a more extensive use of the JSFS classes of polymorphisms for species with high intra-locus recombination rates. First, using a likelihood based method, we demonstrate that taking into account low-frequency polymorphisms shared between species significantly improves the joint estimation of the divergence time and gene flow between species. Second, we introduce a local linear regression algorithm that considerably reduces the computational time and allows for the estimation of unequal rates of gene flow between species. We also investigate which summary statistics from the JSFS allow the greatest estimation accuracy for divergence time and migration rates for low (around 10) and high (around 100) numbers of loci. Focusing on cases with low numbers of loci and high intra-locus recombination rates we show that our methods for the estimation of divergence time and migration rates are more precise than existing approaches.     

Early and late postnatal myocardial and vascular changes in a protein restriction rat model of intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life.     

Activation, regulation, and inhibition of DYRK1A

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase with diverse functions in neuronal development and adult brain physiology. Higher than normal levels of DYRK1A are associated with the pathology of neurodegenerative diseases and have been implicated in some neurobiological alterations of Down syndrome, such as mental retardation. It is therefore important to understand the molecular mechanisms that control the activity of DYRK1A. Here we review the current knowledge about the initial self-activation of DYRK1A by tyrosine autophosphorylation and propose that this mechanism presents an ancestral feature of the CMGC group of kinases. However, tyrosine phosphorylation does not appear to regulate the enzymatic activity of DYRK1A. Control of DYRK1A may take place on the level of gene expression, interaction with regulatory proteins and regulated nuclear translocation. Finally, we compare the properties of small molecule inhibitors that target DYRK1A and evaluate their potential application and limitations. The β-carboline alkaloid harmine is currently the most selective and potent inhibitor of DYRK1A and has proven very useful in cellular assays.     

From cosubstrate similarity to inhibitor diversity--inhibitors of ADP-ribosyltransferases from kinase inhibitor screening

ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.     

2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.     

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.     

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.     

Backbone assignment and secondary structure of the PsbQ protein from Photosystem II

PsbQ is one of the extrinsic proteins situated on the lumenal surface of photosystem II (PSII) in the higher plants and green algae. Its three-dimensional structure was determined by X-ray crystallography with exception of the residues 14–33. To obtain further details about its structure and potentially its dynamics, we approached the problem by NMR. In this paper we report ¹H, ¹⁵N, and ¹³C NMR assignments for the PsbQ protein. The very challenging oligo-proline stretches could be assigned using ¹³C-detected NMR experiments that enabled the assignments of twelve out of the thirteen proline residues of PsbQ. The identification of PsbQ secondary structure elements on the basis of our NMR data was accomplished with the programs TALOS+, web server CS23D and CS-Rosetta. To obtain additional secondary structure information, three-bond H_N-H_α J-coupling constants and deviation of experimental ¹³C_α and ¹³C_β chemical shifts from random coil values were determined. The resulting “consensus” secondary structure of PsbQ compares very well with the resolved regions of the published X-ray crystallographic structure and gives a first estimate of the structure of the “missing link” (i.e. residues 14–33), which will serve as the basis for the further investigation of the structure, dynamics and interactions.