HR‐MAS MRS of the pancreas reveals reduced lipid and elevated lactate and taurine associated with early pancreatic cancer

The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five‐year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS‐detectable metabolic changes provide useful biomarkers for tumor detection and response‐monitoring in other cancers. The goal of this study was to identify MRS‐detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used ¹H high‐resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide‐induced inflammation, in pancreata with pre‐cancerous metaplasia (4 week old p48‐Cre;LSL‐Kras^G12D mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48‐Cre LSL‐Kras^G12D mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre‐cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer‐associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring. Copyright © 2014 John Wiley & Sons, Ltd.     

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.     

Isolation and Characterization of Streptococcus mutans Phage as a Possible Treatment Agent for Caries

Streptococcus mutans is a key bacterium in dental caries, one of the most prevalent chronic infectious diseases. Conventional treatment fails to specifically target the pathogenic bacteria, while tending to eradicate commensal bacteria. Thus, caries remains one of the most common and challenging diseases. Phage therapy, which involves the use of bacterial viruses as anti-bacterial agents, has been gaining interest worldwide. Nevertheless, to date, only a few phages have been isolated against S. mutans. In this study, we describe the isolation and characterization of a new S. mutans phage, termed SMHBZ8, from hundreds of human saliva samples that were collected, filtered, and screened. The SMHBZ8 genome was sequenced and analyzed, visualized by TEM, and its antibacterial properties were evaluated in various states. In addition, we tested the lytic efficacy of SMHBZ8 against S. mutans in a human cariogenic dentin model. The isolation and characterization of SMHBZ8 may be the first step towards developing a potential phage therapy for dental caries.     

Icosapent Ethyl for Primary Versus Secondary Prevention of Major Adverse Cardiovascular Events in Hypertriglyceridemia: Value for Money Analysis

BackgroundIcosapent ethyl (IPE) is approved for the prevention of major adverse cardiovascular events (MACE) in patients with hypertriglyceridemia. However, due to budget constraints, access to IPE will inevitably be limited to a fraction of eligible patients. To help maximize value for money spent, we estimated the number of preventable MACE when providing IPE for primary versus secondary prevention.MethodsThe number of preventable MACE was estimated by dividing the available budget by the cost needed to treat (CNT) to prevent one MACE. CNT was calculated as the product of the number needed to treat (NNT) to prevent 1 MACE by therapy cost. NNT values were determined according to the Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial (REDUCE-IT) results. The budget limit was set as the United States’ threshold suggested by the Institute for Clinical and Economic Review. Sensitivity analysis was performed regarding the cost of IPE in the United States.ResultsThe NNT to prevent 1 MACE over 4.9 years in the Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial primary prevention cohort was 59 (95% confidence interval [CI]: 24-∞) versus 14 (11-21) for secondary prevention. At an annual IPE cost of $2915, the CNT to prevent 1 MACE was $842,726 (95% CI: $342,804-∞) and $199,969 ($157,118-$299,953) accordingly. A total of $819 million worth of IPE can avoid 4762 MACE (95% CI: 0-11,707) versus 20,069 (13,379-25,541), when provided as primary versus secondary prevention therapy; P < .001. The number of avoided MACE is sensitive to IPE price.ConclusionsPrioritizing IPE therapy for patients with an established cardiovascular disease may provide significantly more value for money than primary prevention.     

CHADS2 and CHA2DS2-VASc scores as predictors of platelet reactivity in acute coronary syndrome

BackgroundPlatelet function testing (PFT) in patients treated with P2Y₁₂ inhibitors has been widely evaluated for the prediction of stent thrombosis, myocardial infarction, and bleeding events following percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS). Thus, PFT-guided treatment could positively affect patient outcomes. Data regarding clinical parameters for predicting platelet reactivity in ACS patients are limited. Therefore, our study aims to evaluate CHADS2 and CHA2DS2-VASc scores as predictors for platelet reactivity in ACS patients.MethodsTwo hundred and ninety-one consecutive patients who underwent PCI and were treated with aspirin and clopidogrel due to ACS were tested for their CHADS2, CHA2DS2-VASc scores and platelet reactivity using adenosine diphosphate (ADP)-induced aggregation (conventional aggregometry). Patients were classified into groups according to their CHADS2 and CHA2DS2-VASc scores. Low-risk group (0–1 score) for CHADS2 and CHA2DS2-VASc scores and high-risk group (2–6, 2–9) for CHADS2 and CHA2DS2-VASc scores, respectively. Furthermore, platelet reactivity in each group were compared (low CHADS2 group vs high CHADS2 group, and low CHA2DS2-VASc vs high CHA2DS2-VASc). Platelet reactivity was defined as low platelet reactivity (<19 U), optimal platelet reactivity [(OPR); 19–46 U], and high on-treatment platelet reactivity [(HPR); >46 U]. Thereafter receiver operating characteristic curve analysis was conducted to verify whether CHADS2 and CHA2DS2-VASc scores could predict platelet reactivity.ResultsLow CHADS2 and CHA2DS2-VASc scores were significantly correlated with lower mean platelet ADP-induced aggregation as compared with high CHADS2 and CHA2DS2-VASc scores [45.5 U (± 16) vs. 54.8 U (±15) and 44.2 U (±16) vs. 51.0 U (±17), respectively, p = 0.01 for both].ConclusionIn ACS patients treated with clopidogrel following PCI, high CHADS2 and CHA2DS2-VASc scores correlated with HPR and lower scores correlated with OPR. Further studies are needed to evaluate our findings’ clinical implications.     

Charge of a quasiparticle in a superconductor

Nonlinear charge transport in superconductor–insulator–superconductor (SIS) Josephson junctions has a unique signature in the shuttled charge quantum between the two superconductors. In the zero-bias limit Cooper pairs, each with twice the electron charge, carry the Josephson current. An applied bias V_SD leads to multiple Andreev reflections (MAR), which in the limit of weak tunneling probability should lead to integer multiples of the electron charge ne traversing the junction, with n integer larger than 2Δ/eV_SD and Δ the superconducting order parameter. Exceptionally, just above the gap eV_SD ≥ 2Δ, with Andreev reflections suppressed, one would expect the current to be carried by partitioned quasiparticles, each with energy-dependent charge, being a superposition of an electron and a hole. Using shot-noise measurements in an SIS junction induced in an InAs nanowire (with noise proportional to the partitioned charge), we first observed quantization of the partitioned charge q = e*/e = n, with n = 1–4, thus reaffirming the validity of our charge interpretation. Concentrating next on the bias region eV_SD ～ 2Δ, we found a reproducible and clear dip in the extracted charge to q? ～ 0.6, which, after excluding other possibilities, we attribute to the partitioned quasiparticle charge. Such dip is supported by numerical simulations of our SIS structure.Excitations in superconductors (Bogoliubov quasiparticles) can be described according to the Bardeen–Cooper–Schrieffer (BCS) theory (1) as an energy-dependent superposition of an electron with amplitude u(ε), and a hole with amplitude v(ε), where the energy ε is measured relative to the Fermi energy (2). Evidently, the expectation value of the charge operator (applied to the quasiparticle wave function), which we address as the quasiparticle charge e* = q(ε)e, is smaller than the charge of an electron, q(ε) = |u(ε)|² ? |ν(ε)|² (3). Solving the Bogoliubov–de Gennes equations, one finds that |u(ε)|2=1/2[1+(ε2−Δ2/ε)] and |v(ε)|2=1/2[1−(ε2−Δ2/ε)], with the expected charge evolving with energy according to q(ε)=ε2−Δ2/ε––vanishing altogether at the superconductor gap edges (3). Note, however, that the quasiparticle wave function is not an eigenfunction of the charge operator (3, 4). Properties of quasiparticles, such as the excitation spectra (5), lifetime (6–10), trapping (11), and capturing by Andreev bound states (12, 13), had already been studied extensively; however, studies of their charge are lagging. In the following we present sensitive shot-noise measurements in a Josephson junction, resulting in a clear observation of the quasiparticle charge being smaller than e, q(eV_SD∼2Δ) < 1, and evolving with energy, as expected from the BCS theory.To observe the BCS quasiparticles in transport we study a superconductor–insulator–superconductor (SIS) Josephson junction in the nonlinear regime. The overlap between the wave functions of the quasiparticles in the source and in the drain is expected to result in a tunneling current of their effective charge. This is in contrast with systems which are incoherent (14, 15) or with an isolated superconducting island, where charge conservation leads to traversal of multiples of e – Coulomb charge (16). As current transport in the nonlinear regime results from “multiple Andreev reflections” (MAR), it is prudent to make our measurements credible by first measuring the charge in this familiar regime.In short, the MAR process, described schematically in Fig. 1, carries a signature of the shuttled charge between the two superconductors (SCs), being a consequence of n traversals through the junction (as electron-like and hole-like quasiparticles), with n an integer larger than 2Δ/eV_SD. A low transmission probability t (via tunneling through a barrier) in the bias range 2Δ/n < eV_SD < 2Δ/(n ? 1) assures dominance of the lowest order MAR process (higher orders are suppressed as tⁿ), with the charge evolving in nearly integer multiples of the electron charge. Although there is already a substantial body of theoretical (3, 17–23) and experimental (24–29) studies of the MAR process, charge determination without adjustable parameters is still missing. An important work by Cron et al. (27) indeed showed a staircase-like behavior of the charge using “metallic break junctions;” however, limited sensitivity and the presence of numerous conductance channels some of which with relatively high transmission probabilities did not allow exact charge quantization. Our shot-noise measurements, performed on a quasi-1D Josephson junction (single-mode nanowire) allowed clear observation of charge quantization without adjustable parameters. To count a few advantages: (i) the transmission of the SIS junction could be accurately controlled using a back-gate; (ii) this, along with our high sensitivity in noise measurements, enabled us to pinch the junction strongly (thus suppressing higher MAR orders); and (iii) with the Fermi level located near the 1D channel van Hove singularity, a rather monoenergetic distribution could be injected (SI Appendix, section S7).Open in a separate windowFig. 1.MAR. Illustrations of the leading processes contributing to the current as function of bias. In general, for 2Δ/(n ? 1) > eV_SD > 2Δ/n the leading charge contribution to the current is ne. An electron-like quasiparticle is denoted by a full circle, whereas a hole-like quasiparticle is denoted by an empty circle. (A) When the bias is larger than the energy gap, eV_SD > 2Δ, the leading process is a single-path tunneling of single quasiparticles from the full states (Left) to the empty states (Right). This current is proportional to the transmission coefficient t. Higher-order MAR process (dashed box), being responsible for tunneling of Cooper pairs, is suppressed as t². (B) For 2Δ > eV_SD > Δ, the main charge contributing to the current is 2e with probability t². (C) For Δ > eV_SD > 2Δ/3, the main charge contributing to the current is 3e with probability t³.     

Accuracy of the Global Registry of Acute Coronary Events (GRACE) Risk Score in Contemporary Treatment of Patients With Acute Coronary Syndrome

Background

Global Registry of Acute Coronary Events (GRACE) score has been routinely used for risk stratification in acute coronary syndromes (ACS). We aimed to investigate whether the GRACE score has remained relevant with contemporary treatment of patients with ACS.

Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning “freezes” the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA.