Timeless Links Replication Termination to Mitotic Kinase Activation

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication

Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples — including Xist, which orchestrates X chromosome inactivation — has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Variation of monolayer behaviour and molecular packing in zinc arachidate LB films with subphase pH

Monolayer characteristics and multilayer LB films of zinc arachidate (ZnA) have been studied as a function of subphase pH in the range 4.9–8.0. The monolayer stability was found to be strongly dependent on the subphase pH. Measurement of bilayer spacings in transferred multilayers using X-ray diffraction revealed that depending on subphase pH, three types of molecular packing arrangements corresponding to alkyl chain tilt angles of 0°, 19° and 31° are possible in ZnA multilayers. In certain ranges of subphase pH, more than one type of packing arrangements were found to coexist. These results suggest that the packing arrangement in ZnA multilayers is significantly affected by the properties of the monolayer at the air–water interface and may not be simply related to metal ion electronegativity.© Elsevie

Structure of polymorphic phases in zinc arachidate LB multilayers

The 3D structure of zinc arachidate (ZnA) LB multilayers transferred at different subphase pH has been studied using X-ray scattering and FT-IR spectroscopy. The molecular packing in ZnA multilayers shows an unusually strong dependence on subphase pH, not observed earlier in LB multilayers of divalent fatty acid salts. In all four polymorphic phases, α, β, γ and δ with characteristic 3D structures were observed and in most cases, two or more phases were found to coexist. The δ-phase, which corresponds to the largest alkyl chain tilt angle of not, vert, similar32°, is stable over the complete range of subphase pH investigated and appears as a single phase at a subphase pH of not, vert, similar6.5. It corresponds to a rotator phase like ‘loose packing’ of molecules tilted at an angle of 32° with respect to chain axis, packed in a hexagonal layer cell. The subcell packing changes from hexagonal to orthorhombic and the angle of tilt decreases from 32° (δ-phase) to nearly 0° (α-phase) with increasing pH up to 7.4. The dominant phase at a pH of ~7.4 (α-phase) has a close packed herringbone structure. However at higher subphase pH, the ‘rotator like’ δ-phase regains prominence. The interface morphology of different polymorphic phases is found to be unique, independent of the subphase pH at which the monolayers are transferred.© Elsevie

Structure of CdS–arachidic acid composite LB multilayers

Langmuir–Blodgett (LB) multilayers of cadmium arachidate were used as precursors to grow semiconducting CdS nanoclusters. The formation of CdS in the multilayers was determined by Fourier transform-infrared (FT-IR), ultraviolet–visible (UV–vis) and Raman spectroscopy. The structural changes occurring as a consequence of CdS formation have been characterized using X-ray reflection (XR) and grazing incidence X-ray diffraction (GIXD) techniques. The CdS containing composite multilayers exhibit the presence of two types of molecular domains, one with close packed herringbone arrangement and the other with tilted molecular chains with no in-plane order. The structural and spectroscopic evidences together suggest that the CdS nanoclusters formed within the arachidic acid LB matrix are quasi two-dimensional in nature with lateral dimension ~5–10 nm and thickness ~1.1 nm.© Elsevie

Molecular packing in CdS containing conducting polymer composite LB multilayers

Langmuir–Blodgett (LB) technique has been used to deposit composite multilayers of poly (3-octylthiophene)-cadmium arachidate (POT-CdA) and polyaniline-cadmium arachidate (PANI-CdA). These were used as precursors to develop semiconducting CdS nanoclusters within the conducting polymer based multilayers. The presence of CdS in the multilayers was determined by Fourier transform infrared spectroscopy (FTIR), UV–Vis and Raman spectroscopy. The structural changes occurring as a consequence of CdS formation have been characterized using X-ray reflection and grazing incidence X-ray diffraction (GIXD) techniques. As-deposited POT-CdA multilayers exhibit good vertical as well as in-plane structure similar to that of CdA. In contrast, PANI-CdA multilayers have poor structural order. The in-plane molecular packing in CdA and PANI-CdA multilayers after H2S exposure has mixed domains of rectangular (herringbone) and hexagonal arrangements. In the case of POT-CdA multilayers, however, the original rectangular packing is retained.© Elsevie

Molecular packing in cadmium and zinc arachidate LB multilayers

A combination of grazing incidence X-ray reflection/diffraction (GIXR/GIXD) and atomic force microscopy have been used to study the 3D structure in cadmium arachidate (CdA) and single phase zinc arachidate (ZnA) LB multilayers. The CdA multilayers have an ideal close packed herringbone structure. The molecules have a non-centred in-plane arrangement with specific orientational relation between the central and the corner molecules. In contrast, the ZnA multilayers have a hexagonal layer plane packing with tilted molecules. The molecules are loosely packed with rotational freedom about chain axis as observed in ‘rotator’ phases.© Elsevie