The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ～10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

Diversity and partitioning of bacterial populations within the accessory nidamental gland of the squid Euprymna scolopes

Microbial consortia confer important benefits to animal and plant hosts, and model associations are necessary to examine these types of host/microbe interactions. The accessory nidamental gland (ANG) is a female reproductive organ found among cephalopod mollusks that contains a consortium of bacteria, the exact function of which is unknown. To begin to understand the role of this organ, the bacterial consortium was characterized in the Hawaiian bobtail squid, Euprymna scolopes, a well-studied model organism for symbiosis research. Transmission electron microscopy (TEM) analysis of the ANG revealed dense bacterial assemblages of rod- and coccus-shaped cells segregated by morphology into separate, epithelium-lined tubules. The host epithelium was morphologically heterogeneous, containing ciliated and nonciliated cells with various brush border thicknesses. Hemocytes of the host's innate immune system were also found in close proximity to the bacteria within the tubules. A census of 16S rRNA genes suggested that Rhodobacterales, Rhizobiales, and Verrucomicrobia bacteria were prevalent, with members of the genus Phaeobacter dominating the consortium. Analysis of 454-shotgun sequencing data confirmed the presence of members of these taxa and revealed members of a fourth, Flavobacteria of the Bacteroidetes phylum. 16S rRNA fluorescent in situ hybridization (FISH) revealed that many ANG tubules were dominated by members of specific taxa, namely, Rhodobacterales, Verrucomicrobia, or Cytophaga-Flavobacteria-Bacteroidetes, suggesting symbiont partitioning to specific host tubules. In addition, FISH revealed that bacteria, including Phaeobacter species from the ANG, are likely deposited into the jelly coat of freshly laid eggs. This report establishes the ANG of the invertebrate E. scolopes as a model to examine interactions between a bacterial consortium and its host.     

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.     

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ～6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.     

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase

Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90?? in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.     

[Zn(phen)(O,N,O)(H2O)] and [Zn(phen)(O,N)(H2O)] with O,N,O?is?2,6-dipicolinate and N,O?is?l-threoninate: synthesis, characterization, and biomedical properties

Two ternary Zn(II) complexes, with 1,10-phenanthroline (phen) as the main ligand and a carboxylate-containing ligand [dipicolinate (dipico) or L-threoninate (L-Thr)] as the subsidiary ligand, were prepared and characterized by elemental analysis, Fourier transform IR, UV, and fluorescence spectroscopy, X-ray diffraction, molar conductivity, and electrospray ionization mass spectrometry. X-ray structure analysis shows that both [Zn(phen)(dipico)(H(2)O)]·H(2)O (1) and [Zn(phen)(L-Thr)(H(2)O)Cl]·2H(2)O (2) have octahedral geometry about the Zn(II) atom. Both complexes can inhibit topoisomerase I, and have better anticancer activity than cisplatin against nasopharyngeal cancer cell lines, HK1 and HONE-1, with concentrations causing 50?% inhibition of cell proliferation (IC(50)) in the low micromolar range. Complex 2 has the highest therapeutic index for HK1. Both Zn(II) complexes can induce cell death by apoptosis. Changing the subsidiary ligand in the Zn(II) complexes affects the UV-fluorescence spectral properties of the coordinated phen ligand, the binding affinity for some DNA sequences, nucleobase sequence-selective binding, the phase at which cell cycle progression was arrested for treated cancer cells, and their therapeutic index.     

CMOS-compatible, label-free silicon-nanowire biosensors to detect cardiac troponin I for acute myocardial infarction diagnosis

A label-free biosensor for electrical detection of cardiac troponin I (cTnI), a highly sensitive and selective biomarker of acute myocardial infarction (AMI), is demonstrated using silicon nanowire (SiNW) based field-effect transistors (FETs). The FET devices were fabricated by a complementary metal oxide semiconductor (CMOS) compatible top-down approach to define the SiNW followed by tetramethylammonium hydroxide (TMAH) wet etching. Electrical characterizations of the SiNW FET revealed an ambipolar conduction characteristic with an on/off ratio of 10(5)-10(6). CTnI monoclonal antibodies were then covalently immobilized on the SiNW surfaces. By integrating with a homemade biosensor measurement system, the biosensor exhibited rapid and sensitive response to cTnI proteins. The current response showed a nature of logarithm relationship against the cTnI concentration from 46 ng/mL down to 0.092 ng/mL. Moreover, an anti-interference capability of the fabricated biosensor was also assessed. By utilizing the top-down fabrication method, this work provides an efficient way for the cTnI proteins detection with an enormous potential of mass-production, which definitely facilitate the practical applications.