Crystal Structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3 ' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a 5 ' end binding pocket that hosts the 5 ' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3 ' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5 ' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3 ' flap DNA substrates with 5 ' nicked ends.X11119Ysciescopu

Tolfenamic Acid Induces Apoptosis and Growth Inhibition in Head and Neck Cancer: Involvement of NAG-1 Expression

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is induced by nonsteroidal anti-inflammatory drugs and possesses proapoptotic and antitumorigenic activities. Although tolfenamic acid (TA) induces apoptosis in head and neck cancer cells, the relationship between NAG-1 and TA has not been determined. This study investigated the induction of apoptosis in head and neck cancer cells treated by TA and the role of NAG-1 expression in this induction. TA reduced head and neck cancer cell viability in a dose-dependent manner and induced apoptosis. The induced apoptosis was coincident with the expression of NAG-1. Overexpression of NAG-1 enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. TA significantly inhibited tumor formation as assessed by xenograft models, and this result accompanied the induction of apoptotic cells and NAG-1 expression in tumor tissue samples. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression in head and neck squamous cell carcinoma, providing an additional mechanistic explanation for the apoptotic activity of TA

Edge-Functionalization of Pyrene as a Miniature Graphene via Friedel–Crafts Acylation Reaction in Poly(Phosphoric Acid)

The feasibility of edge-functionalization of graphite was tested via the model reaction between pyrene and 4-(2,4,6-trimethylphenyloxy)benzamide (TMPBA) in poly(phosphoric acid) (PPA)/phosphorous pentoxide (P2O5) medium. The functionalization was confirmed by various characterization techniques. On the basis of the model study, the reaction condition could be extended to the edge-functionalization of graphite with TMPBA. Preliminary results showed that the resultant TMPBA-grafted graphite (graphite-g-TMPBA) was found to be readily dispersible in N-methyl-2-pyrrolidone (NMP) and can be used as a precursor for edge-functionalized graphene (EFG)

Flavor Physics in an SO(10) Grand Unified Model

In supersymmetric grand-unified models, the lepton mixing matrix can possibly affect flavor-changing transitions in the quark sector. We present a detailed analysis of a model proposed by Chang, Masiero and Murayama, in which the near-maximal atmospheric neutrino mixing angle governs large new b -> s transitions. Relating the supersymmetric low-energy parameters to seven new parameters of this SO(10) GUT model, we perform a correlated study of several flavor-changing neutral current (FCNC) processes. We find the current bound on B(tau -> mu gamma) more constraining than B(B -> X_s gamma). The LEP limit on the lightest Higgs boson mass implies an important lower bound on tan beta, which in turn limits the size of the new FCNC transitions. Remarkably, the combined analysis does not rule out large effects in B_s-B_s-bar mixing and we can easily accomodate the large CP phase in the B_s-B_s-bar system which has recently been inferred from a global analysis of CDF and DO data. The model predicts a particle spectrum which is different from the popular Constrained Minimal Supersymmetric Standard Model (CMSSM). B(tau -> mu gamma) enforces heavy masses, typically above 1 TeV, for the sfermions of the degenerate first two generations. However, the ratio of the third-generation and first-generation sfermion masses is smaller than in the CMSSM and a (dominantly right-handed) stop with mass below 500 GeV is possible.Comment: 44 pages, 5 figures. Footnote and references added, minor changes, Fig. 2 corrected; journal versio

Lrp Acts as Both a Positive and Negative Regulator for Type 1 Fimbriae Production in Salmonella enterica Serovar Typhimurium

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment

Two loop electroweak corrections to Bˉ→Xsγ\bar B\rightarrow X_s\gamma and Bs0→μ+μ−B_s^0\rightarrow \mu^+\mu^- in the B-LSSM

The rare decays $\bar B\rightarrow X_s\gamma$ and $B_s^0\rightarrow \mu^+\mu^-$ are important to research new physics beyond standard model. In this work, we investigate two loop electroweak corrections to $\bar B\rightarrow X_s\gamma$ and $B_s^0\rightarrow \mu^+\mu^-$ in the minimal supersymmetric extension of the SM with local $B-L$ gauge symmetry (B-LSSM), under a minimal flavor violating assumption for the soft breaking terms. In this framework, new particles and new definition of squarks can affect the theoretical predictions of these two processes, with respect to the MSSM. Considering the constraints from updated experimental data, the numerical results show that the B-LSSM can fit the experimental data for the branching ratios of $\bar B\rightarrow X_s\gamma$ and $B_s^0\rightarrow \mu^+\mu^-$. The results of the rare decays also further constrain the parameter space of the B-LSSM.Comment: 33 pages, 9 figures, Published in EPJ

Lepton flavour violation in the MSSM

We derive new constraints on the quantities delta_{XY}^{ij}, X,Y=L,R, which parametrise the flavour-off-diagonal terms of the charged slepton mass matrix in the MSSM. Considering mass and anomalous magnetic moment of the electron we obtain the bound |delta^{13}_{LL} delta^{13}_{RR}|<0.1 for tan beta=50, which involves the poorly constrained element delta^{13}_{RR}. We improve the predictions for the decays tau -> mu gamma, tau -> e gamma and mu -> e gamma by including two-loop corrections which are enhanced if tan beta is large. The finite renormalisation of the PMNS matrix from soft SUSY-breaking terms is derived and applied to the charged-Higgs-lepton vertex. We find that the experimental bound on BR(tau -> e gamma) severely limits the size of the MSSM loop correction to the PMNS element U_{e3}, which is important for the proper interpretation of a future U_{e3} measurement. Subsequently we confront our new values for delta^{ij}_{LL} with a GUT analysis. Further, we include the effects of dimension-5 Yukawa terms, which are needed to fix the Yukawa unification of the first two generations. If universal supersymmetry breaking occurs above the GUT scale, we find the flavour structure of the dimension-5 Yukawa couplings tightly constrained by mu -> e gamma.Comment: 37 pages, 15 figures; typo in Equation (35) and (49) correcte

Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation

Funding: Investigator Award from the Wellcome Trust (098391/Z/12/Z) and (217196/Z/19/Z) and a Programme grant from Cancer Research UK (C434/A21747) to R.T.H.; J.C.P. thanks the University of St Andrews for financial support.Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.Publisher PDFPeer reviewe

LAP2 Is Widely Overexpressed in Diverse Digestive Tract Cancers and Regulates Motility of Cancer Cells

BACKGROUND: Lamina-associated polypeptides 2 (LAP2) is a nuclear protein that connects the nuclear lamina with chromatin. Although its critical roles in genetic disorders and hematopoietic malignancies have been described, its expression and roles in digestive tract cancers have been poorly characterized. METHODS: To examine the expression of LAP2 in patient tissues, we performed immunohistochemistry and real-time PCR. To examine motility of cancer cells, we employed Boyden chamber, wound healing and Matrigel invasion assays. To reveal its roles in metastasis in vivo, we used a liver metastasis xenograft model. To investigate the underlying mechanism, a cDNA microarray was conducted. RESULTS: Immunohistochemistry in patient tissues showed widespread expression of LAP2 in diverse digestive tract cancers including stomach, pancreas, liver, and bile duct cancers. Real-time PCR confirmed that LAP2β is over-expressed in gastric cancer tissues. Knockdown of LAP2β did not affect proliferation of most digestive tract cancer cells except pancreatic cancer cells. However, knockdown of LAP2β decreased motility of all tested cancer cells. Moreover, overexpression of LAP2β increased motility of gastric and pancreatic cancer cells. In the liver metastasis xenograft model, LAP2β increased metastatic efficacy of gastric cancer cells and mortality in tested mice. cDNA microarrays showed the possibility that myristoylated alanine-rich C kinase substrate (MARCKS) and interleukin6 (IL6) may mediate LAP2β-regulated motility of cancer cells. CONCLUSIONS: From the above results, we conclude that LAP2 is widely overexpressed in diverse digestive tract cancers and LAP2β regulates motility of cancer cells and suggest that LAP2β may have utility for diagnostics and therapeutics in digestive tract cancers

The Marker State Space (MSS) Method for Classifying Clinical Samples

The development of accurate clinical biomarkers has been challenging in part due to the diversity between patients and diseases. One approach to account for the diversity is to use multiple markers to classify patients, based on the concept that each individual marker contributes information from its respective subclass of patients. Here we present a new strategy for developing biomarker panels that accounts for completely distinct patient subclasses. Marker State Space (MSS) defines "marker states" based on all possible patterns of high and low values among a panel of markers. Each marker state is defined as either a case state or a control state, and a sample is classified as case or control based on the state it occupies. MSS was used to define multi-marker panels that were robust in cross validation and training-set/test-set analyses and that yielded similar classification accuracy to several other classification algorithms. A three-marker panel for discriminating pancreatic cancer patients from control subjects revealed subclasses of patients based on distinct marker states. MSS provides a straightforward approach for modeling highly divergent subclasses of patients, which may be adaptable for diverse applications. © 2013 Fallon et al