Time-scale and other invariants of integrative mechanical behavior in living cells.

In dealing with systems as complex as the cytoskeleton, we need organizing principles or, short of that, an empirical framework into which these systems fit. We report here unexpected invariants of cytoskeletal behavior that comprise such an empirical framework. We measured elastic and frictional moduli of a variety of cell types over a wide range of time scales and using a variety of biological interventions. In all instances elastic stresses dominated at frequencies below 300 Hz, increased only weakly with frequency, and followed a power law; no characteristic time scale was evident. Frictional stresses paralleled the elastic behavior at frequencies below 10 Hz but approached a Newtonian viscous behavior at higher frequencies. Surprisingly, all data could be collapsed onto master curves, the existence of which implies that elastic and frictional stresses share a common underlying mechanism. Taken together, these findings define an unanticipated integrative framework for studying protein interactions within the complex microenvironment of the cell body, and appear to set limits on what can be predicted about integrated mechanical behavior of the matrix based solely on cytoskeletal constituents considered in isolation. Moreover, these observations are consistent with the hypothesis that the cytoskeleton of the living cell behaves as a soft glassy material, wherein cytoskeletal proteins modulate cell mechanical properties mainly by changing an effective temperature of the cytoskeletal matrix. If so, then the effective temperature becomes an easily quantified determinant of the ability of the cytoskeleton to deform, flow, and reorganize

Global comparison of warring groups in 2002–2007: fatalities from targeting civilians vs. fighting battles

Background Warring groups that compete to dominate a civilian population confront contending behavioral options: target civilians or battle the enemy. We aimed to describe degrees to which combatant groups concentrated lethal behavior into intentionally targeting civilians as opposed to engaging in battle with opponents in contemporary armed conflict. Methodology/Principal Findings We identified all 226 formally organized state and non-state groups (i.e. actors) that engaged in lethal armed conflict during 2002–2007: 43 state and 183 non-state. We summed civilians killed by an actor's intentional targeting with civilians and combatants killed in battles in which the actor was involved for total fatalities associated with each actor, indicating overall scale of armed conflict. We used a Civilian Targeting Index (CTI), defined as the proportion of total fatalities caused by intentional targeting of civilians, to measure the concentration of lethal behavior into civilian targeting. We report actor-specific findings and four significant trends: 1.) 61% of all 226 actors (95% CI 55% to 67%) refrained from targeting civilians. 2.) Logistic regression showed actors were more likely to have targeted civilians if conflict duration was three or more years rather than one year. 3.) In the 88 actors that targeted civilians, multiple regressions showed an inverse correlation between CTI values and the total number of fatalities. Conflict duration of three or more years was associated with lower CTI values than conflict duration of one year. 4.) When conflict scale and duration were accounted for, state and non-state actors did not differ. We describe civilian targeting by actors in prolonged conflict. We discuss comparable patterns found in nature and interdisciplinary research. Conclusions/Significance Most warring groups in 2002–2007 did not target civilians. Warring groups that targeted civilians in small-scale, brief conflict concentrated more lethal behavior into targeting civilians, and less into battles, than groups in larger-scale, longer conflict

Quantitative Analysis of Immune Infiltrates in Primary Melanoma.

Novel methods to analyze the tumor microenvironment (TME) are urgently needed to stratify melanoma patients for adjuvant immunotherapy. Tumor-infiltrating lymphocyte (TIL) analysis, by conventional pathologic methods, is predictive but is insufficiently precise for clinical application. Quantitative multiplex immunofluorescence (qmIF) allows for evaluation of the TME using multiparameter phenotyping, tissue segmentation, and quantitative spatial analysis (qSA). Given that CD3+CD8+ cytotoxic lymphocytes (CTLs) promote antitumor immunity, whereas CD68+ macrophages impair immunity, we hypothesized that quantification and spatial analysis of macrophages and CTLs would correlate with clinical outcome. We applied qmIF to 104 primary stage II to III melanoma tumors and found that CTLs were closer in proximity to activated (CD68+HLA-DR+) macrophages than nonactivated (CD68+HLA-DR-) macrophages (P < 0.0001). CTLs were further in proximity from proliferating SOX10+ melanoma cells than nonproliferating ones (P < 0.0001). In 64 patients with known cause of death, we found that high CTL and low macrophage density in the stroma (P = 0.0038 and P = 0.0006, respectively) correlated with disease-specific survival (DSS), but the correlation was less significant for CTL and macrophage density in the tumor (P = 0.0147 and P = 0.0426, respectively). DSS correlation was strongest for stromal HLA-DR+ CTLs (P = 0.0005). CTL distance to HLA-DR- macrophages associated with poor DSS (P = 0.0016), whereas distance to Ki67- tumor cells associated inversely with DSS (P = 0.0006). A low CTL/macrophage ratio in the stroma conferred a hazard ratio (HR) of 3.719 for death from melanoma and correlated with shortened overall survival (OS) in the complete 104 patient cohort by Cox analysis (P = 0.009) and merits further development as a biomarker for clinical application

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency

Violent Deaths of Iraqi Civilians, 2003–2008: Analysis by Perpetrator, Weapon, Time, and Location

Madelyn Hsiao-Rei Hicks and colleagues provide a detailed analysis of Iraqi civilian violent deaths during 2003-2008 of the Iraq war and show that of 92,614 deaths, unknown perpetrators caused 74% of deaths, Coalition forces 12%, and Anti-Coalition forces 11%

Detection of erbB2 copy number variations in plasma of patients with esophageal carcinoma

<p>Abstract</p> <p>Background</p> <p>Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression.</p> <p>Methods</p> <p>Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the <it>erbB2 </it>gene using real-time PCR assays.</p> <p>Results</p> <p>The real-time PCR assays for <it>erbB2 </it>gene showed significant (<it>P </it>= 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in <it>erbB2 </it>were negatively correlated to the progression free survival of these patients (<it>P </it>= 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels.</p> <p>Conclusion</p> <p>The copy number variation of <it>erbB2 </it>gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer.</p

Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes

Type 1 diabetes (T1D) is a common autoimmune disorder that arises from the action of multiple genetic and environmental risk factors. We report the findings of a genome-wide association study of T1D, combined in a meta-analysis with two previously published studies. The total sample set included 7,514 cases and 9,045 reference samples. Forty-one distinct genomic locations provided evidence for association with T1D in the meta-analysis (P 10 6). After excluding previously reported associations, we further tested 27 regions in an independent set of 4,267 cases, 4,463 controls and 2,319 affected sib-pair (ASP) families. Of these, 18 regions were replicated (P 0.01; overall P 5 × 10 8) and 4 additional regions provided nominal evidence of replication (P 0.05). The many new candidate genes suggested by these results include IL10, IL19, IL20, GLIS3, CD69 and IL27