Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells.

In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.

Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 µM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

Impact of Nano- and Micro-Sized Chromium(III) Particles on Cytotoxicity and Gene Expression Profiles Related to Genomic Stability in Human Keratinocytes and Alveolar Epithelial Cells.

Exposure to Cr(VI) compounds has been consistently associated with genotoxicity and carcinogenicity, whereas Cr(III) is far less toxic, due to its poor cellular uptake. However, contradictory results have been published in relation to particulate Cr2O3. The aim of the present study was to investigate whether Cr(III) particles exerted properties comparable to water soluble Cr(III) or to Cr(VI), including two nano-sized and one micro-sized particles. The morphology and size distribution were determined by TEM, while the oxidation state was analyzed by XPS. Chromium release was quantified via AAS, and colorimetrically differentiated between Cr(VI) and Cr(III). Furthermore, the toxicological fingerprints of the Cr2O3 particles were established using high-throughput RT-qPCR and then compared to water-soluble Cr(VI) and Cr(III) in A549 and HaCaT cells. Regarding the Cr2O3 particles, two out of three exerted only minor or no toxicity, and the gene expression profiles were comparable to Cr(III). However, one particle under investigation released considerable amounts of Cr(VI), and also resembled the toxicity profiles of Cr(VI); this was also evident in the altered gene expression related to DNA damage signaling, oxidative stress response, inflammation, and cell death pathways. Even though the highest toxicity was found in the case of the smallest particle, size did not appear to be the decisive parameter, but rather the purity of the Cr(III) particles with respect to Cr(VI) content.

Preconcentration of f-elements from aqueous solution utilizing a modified carbon paste electrode.

An evaluation using paraffin oil based, Acheson 38 carbon paste electrodes modified with α-hydroxyisobutyric acid (HIBA) to preconcentrate f-elements cathodically is described. The modified paste was made by directly mixing solid HIBA into the carbon paste. A chemically reversible cyclic voltammogram for HIBA was observed on this modified carbon paste, which was found to be a non-Nerstian, single electron transfer process. Lanthanides (less promethium) were found to accumulate onto the electrode surface during a 30 s electrodeposition step at -0.4 V vs Ag/AgCl from 0.1 M LiCl. The elements were then stripped off into a 2% HNO(3) solution by an oxidative step at +0.8 V vs Ag/AgCl; quantitative removal from the electrode was confirmed by ICPMS. Ultratrace solutions with initial concentrations down to 5 parts per quadrillion (ppq) were preconcentrated in 5 min above our instrumental limit of detection (LOD) of around 1 ppt for lanthanides.

Cathodic preconcentration of f-elements on a mercury film carbon fiber disk microelectrode.

Field detection and quantification of f-elements is an important problem in radioanalytical chemistry requiring small, portable devices. Here, characterization of a 10 µm Hg film carbon fiber disk microelectrode to accumulate f-elements is described. Accumulation was performed by cathodic deposition and evaluated by anodic stripping and subsequent ICPMS analyses. La(3+) was used as the model element, and subsequent studies were conducted on a 17 element mixture (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Th). In the model studies, La(3+) undergoes a sorption phenomenon, and as in other studies and confirmed by ICPMS, a monolayer of atoms on the electrode surface is formed. Dissolved O(2) was found to have no effect on the cathodic accumulation of La(3+). Consideration of electrode reaction conditions is made, and reactions are hypothesized. The limit of detection (LOD) was found to be 10(-7) M with mass detection of 10(9) atoms, approximately 5 orders of magnitude less than at conventionally sized electrodes. To solve a dilution problem in follow-on analyses, a suggestion to use microelectrode chip-based sensors was made.

Comparison of Metal-Based Nanoparticles and Nanowires: Solubility, Reactivity, Bioavailability and Cellular Toxicity.

While the toxicity of metal-based nanoparticles (NP) has been investigated in an increasing number of studies, little is known about metal-based fibrous materials, so-called nanowires (NWs). Within the present study, the physico-chemical properties of particulate and fibrous nanomaterials based on Cu, CuO, Ni, and Ag as well as TiO2 and CeO2 NP were characterized and compared with respect to abiotic metal ion release in different physiologically relevant media as well as acellular reactivity. While none of the materials was soluble at neutral pH in artificial alveolar fluid (AAF), Cu, CuO, and Ni-based materials displayed distinct dissolution under the acidic conditions found in artificial lysosomal fluids (ALF and PSF). Subsequently, four different cell lines were applied to compare cytotoxicity as well as intracellular metal ion release in the cytoplasm and nucleus. Both cytotoxicity and bioavailability reflected the acellular dissolution rates in physiological lysosomal media (pH 4.5); only Ag-based materials showed no or very low acellular solubility, but pronounced intracellular bioavailability and cytotoxicity, leading to particularly high concentrations in the nucleus. In conclusion, in spite of some quantitative differences, the intracellular bioavailability as well as toxicity is mostly driven by the respective metal and is less modulated by the shape of the respective NP or NW.

Cardiac MRI shows an association of lower cardiorespiratory fitness with decreased myocardial mass and higher cardiac stiffness in the general population - The Sedentary's Heart.

BACKGROUND: The heart has the capacity to adapt to different demands. The pathophysiological mechanisms involved with sedentarism are not fundamentally the opposite of those related with physical activity and regular exercise. We investigated the impact of lower cardiorespiratory fitness (CRF) on heart's plasticity and function in a population-based setting. METHODS: We used data from 1165 participants (539 women; 46.3%) aged 21-81 years from two independent cohorts of the Study of Health in Pomerania (SHIP-2 and SHIP-TREND-0). We analyzed the cross-sectional associations of peak oxygen uptake (VO2peak), determined by symptom-limited cardiopulmonary exercise testing, with structural and functional left ventricular (LV) and left atrial (LA) parameters determined by magnetic resonance imaging (MRI) using multivariable- adjusted linear regression models. RESULTS: A 1 L/min lower VO2peak was associated with a 10.5 g (95% confidence interval: 8.00 to 12.9; p < 0.001) lower LV mass, a 14.8 mL (10.9 to 18.6; p < 0.001) lower LV end-diastolic volume, a 0.29 mm (0.19 to 0.40; p < 0.001) lower LV wall-thickness, a 8.85 mL/beat (6.53 to 11.2; p < 0.001) lower LV stroke volume, a 0.42 L/min (0.25 to 0.60; p < 0.001) lower LV cardiac output and a 7.51 mL (3.88 to 11.1; p < 0.001) lower LA end-diastolic volume. Moreover, there were no associations with a concentric or eccentric remodeling and LV and LA ejection fraction. CONCLUSIONS: Lower CRF was associated with a smaller heart, LV wall-thickness and mass, LV and LA stroke volume and cardiac output. Conversely, there was no association with LA and LV ejection fraction. Our cross-sectional observations are consistent with cardiac adaptations reflecting reduced volume loading demands of a sedentary lifestyle - "the sedentary's heart".

Preconcentration of trivalent lanthanide elements on a mercury film from aqueous solution using rotating disk electrode voltammetry.

An approach to concentrate trivalent lanthanide elements into or onto mercury film electrodes supported on rotating disk glassy carbon electrodes in small volumes (

Vacuum-pack temporary abdominal wound management with delayed-closure for the management of ruptured abdominal aortic aneurysm and other abdominal vascular catastrophes: absence of graft infection in long-term survivors.

Patients who undergo open repair of ruptured abdominal aortic aneurysms (rAAA) may require delayed abdominal wound closure to prevent the adverse consequences of intra-abdominal hypertension and abdominal compartment syndrome. However, surgeons may be reticent to use delayed abdominal closure techniques due to concern that such management may increase the risk of graft infection. We retrospectively reviewed our patient experience with rAAA and other vascular catastrophes in which vacuum-pack abdominal wound management with delayed closure was used between 2000 and 2007. Eighteen of 23 patients treated with delayed closure survived (78%). In five early deaths, graft infection was not clinically suspected. Sixteen of 20 rAAA patients survived, with abdominal wound closure achieved at a mean of 4 days. Mean follow up was 53 months, the longest among comparable series. Graft infection has not been encountered in this group. Our results suggest that the potential risk of graft infection should not outweigh the benefits of vacuum-pack temporary abdominal wound management with delayed closure in critically ill patients at high risk for intra-abdominal hypertension and abdominal compartment syndrome after major abdominal vascular procedures.

Endovenous stent-assisted coil embolization for a symptomatic femoral vein aneurysm.

An 83-year old man presented with recurrent pulmonary embolism originating from a distal left superficial femoral vein aneurysm despite therapeutic anticoagulation. We treated the patient transluminally using the technique of stent-assisted coil embolization via percutaneous transpopliteal venous access. Follow-up by serial duplex ultrasonography and computer tomographic venography (CTV) demonstrated resolution of the aneurysm. Our case demonstrates that stent-assisted coil embolization may effectively exclude a saccular venous aneurysm and prevent recurrent pulmonary embolization.

Monocyte-platelet aggregates and CD11b expression as markers for thrombogenicity in atrial fibrillation.

BACKGROUND: A strong interdependence is known between atrial fibrillation (AF), inflammation and thrombogenesis. Monocyte-platelet aggregates (MPAs) are sensitive markers of platelets and monocyte activation. It is not known whether MPAs are associated with thrombogenicity in AF. Therefore, we examined differences in the content of MPAs and CD11b expression in patients with AF in dependence of the presence of atrial thrombus formation. METHODS: 107 patients with symptomatic AF underwent transesophageal echocardiography (TEE) before planned cardioversion or pulmonary vein isolation. Flow-cytometric quantification analysis was done on the day of performed TEE to determine the content of MPAs and the expression of CD11b on monocytes and granulocytes. RESULTS: Compared to patients without thrombus (n = 80) those with an echocardiographic proven left atrium (LA) thrombus (n = 27) showed an increased extent of the risk factors age, diabetes and heart failure. The content of MPAs (147 ± 12 vs. 311 ± 29 cells/µl, p < 0.001) as well as the CD11b expression on monocytes (p < 0.05) and granulocytes (p < 0.05) were strongly associated with the existence of a LA thrombus. The content of MPAs and the CD11b expression remained independent predictors for LA thrombus after adjustment in logistic regression analysis and negatively correlated with left atrial appendage flow velocity. MPAs above 170 cells/µl (OR 34.2, p = 0.01) had a sensitivity of 96 % and a specificity of 73 % for predicting LA-thrombus. CONCLUSIONS: The content of MPAs and the CD11b expression on monocytes and granulocytes are increased in AF-patients with proven thrombus formation. They seem to be appropriate biomarkers for stratification of thromboembolic risk in patients with AF.

What can be learned from studies of multisubstrate mechanisms of neuronal dopamine transport?

The dopamine transporter (DAT) is a Na+- and Cl--dependent transporter and, with respect to its three apparent substrates, both partially random sequential as well as ordered mechanisms have been reported. Here we describe some of the features of DAT, such as the coupling of energy to concentrate dopamine and the properties of slippage and leakage. Further, in considering the regulation of transport velocities by DAT few have considered issues related to substrate regulation of DAT activity. Specifically, what effect do changes in the constants (K) for the participation of Na+ and Cl- have on dopamine transport velocity? It is shown that DAT may possess properties of slippage, an argument is made that leakage may be important in neuronal systems containing DAT, and the influence of changing values of K for the participation of Na+ and Cl- in transport is shown to produce large effects on DAT activity depending on the multisubstrate kinetic mechanism.

Retrospective analysis of clinical and laboratory factors associated with lymphoma in simian AIDS.

A nonhuman primate model for AIDS-associated Non-Hodgkin's lymphoma (AIDS-NHL) has been described in which animals inoculated with simian immunodeficiency virus (SIV) develop simian AIDS (SAIDS) and SAIDS-NHL. The objective of the present study was to describe statistically the major trends observed in clinical and laboratory data collected longitudinally on a large cohort of nonhuman primates that developed SAIDS-NHL. Clinical and laboratory data were collected longitudinally on each animal from the time of SIV infection throughout progression to lymphoma. Data were analyzed retrospectively with regard to species, gender, age at SIV inoculation, survival, cause of death, CD4+ T-cell and B-cell counts, SIV antigenemia, persistent lymphoid hyperplasia and lymphocryptovirus infection. Median survival time (354 days: 95% CI 309-388) was not related to gender, age at SIV inoculation, cause of death, or RhLCV infection. Survival was not related to CD4+ T-cell count at the time of SIV infection (P = 0.5531), but increased survival was significantly related to a slower rate of CD4+ T-cell decline (P = 0.0256). A B-cell expansion was observed at the midpoint of disease. A steep rise in SIV antigenemia was detected in the first 21 days of infection followed by a rapid decline. This pattern did not occur in animals inoculated with SIV as infants or yearlings. Of 45 cases, 9 exhibited marked, persistent lymphoid hyperplasia. These results describe trends identified in clinical and laboratory factors associated with SAIDS-NHL in the largest collection of such samples in the world. The results contribute to an understanding of the etiology of SAIDS-NHL and to the future development of useful predictors of SAIDS- or AIDS-related lymphoma.

Tibial artery calcification as a marker of amputation risk in patients with peripheral arterial disease.

OBJECTIVES: The purpose of this study was to evaluate the relationship between calcification in tibial arteries, the degree of limb ischemia, and the near-term risk of amputation. BACKGROUND: Determining the amputation risk in patients with peripheral arterial disease (PAD) remains difficult. Developing new measures to identify patients who are at high risk for amputation would allow for targeted interventions and focused trials aimed at limb preservation. METHODS: Two hundred twenty-nine patients underwent evaluation by history, arterial Doppler, and multislice computed tomography of the lower extremities. We then explored the relationship between a tibial artery calcification (TAC), traditional risk factors for PAD, limb status at presentation, and near-term amputation risk. RESULTS: Increased age and traditional atherosclerosis risk factors were associated with higher TAC scores. Patients with critical limb ischemia had the highest TAC scores, and increasing TAC scores were associated with worsening levels of limb ischemia in ordinal regression analysis. Receiver-operator characteristic analysis suggested that the TAC score predicted amputation better than the ankle-brachial index (ABI). Symptomatic patients with a TAC score greater than 400 had a significantly increased risk of amputation. In Cox regression analysis, there was a strong association between the TAC score and the risk of major amputation that remained after adjustment for traditional risk factors and the ABI. CONCLUSIONS: In patients presenting with PAD, the TAC score is associated with the stage of disease and it identifies those who are at high risk for amputation better than traditional risk factors and an abnormal ABI.

The metastatic lymph node ratio predicts survival in colon cancer.

BACKGROUND: Lymph node metastases are the most important predictor of survival in non-stage IV colon cancer. Recent studies of gastric cancer have shown a prognostic significance of a lymph node ratio (number of positive nodes divided by total number harvested). Our goal was to determine whether a lymph node ratio (LNR) would predict disease-free survival (DFS) and overall survival (OS) in a tertiary care Veterans Affairs medical center. METHODS: A retrospective review of a prospectively collected database of colon cancer patients was performed to determine the effect of LNR on DFS and OS. A cohort of 232 patients with non-stage IV colon cancer was eligible for analysis. Survival curves were constructed using the Kaplan-Meier method. RESULTS: For all patients, a LNR of .08 was identified as a breakpoint for predicting OS and DFS. Specific analysis of stage III patients revealed that a LNR of .18 was predictive of DFS. CONCLUSIONS: This study showed the prognostic significance of ratio-based staging for colon cancer and may aid in future staging systems.

Ischemic complications of percutaneous femoral artery catheterization.

Ischemic injuries following percutaneous femoral artery catheterization are uncommon but have been associated with vascular closure devices (VCDs). The purpose of this study was to retrospectively compare ischemic and hemorrhagic complications of femoral artery catheterization and to identify factors associated with ischemic injuries. The operative registries of the attending vascular surgeons at one academic and two community hospitals were retrospectively reviewed to identify all complications of femoral artery catheterization requiring operative intervention. Demographic, clinical, procedural, operative, and outcome data were compared between patients who sustained ischemic and hemorrhagic complications. From January 2001 to December 2006, 95 patients required operative management of complications related to femoral artery catheterization including 40 patients who experienced ischemic (group 1) and 55 patients who experienced hemorrhagic (group 2) complications. Compared to those sustaining hemorrhagic complications, ischemic complications were more frequently associated with younger age, smoking, VCD deployment, and, when controlling for VCD use, female gender. Time to presentation was also significantly longer in patients experiencing ischemic complications. Ischemic complications are increasingly recognized following femoral artery catheterization. Vascular surgeons should anticipate a new pattern of injury following femoral artery catheterization, one that often requires complex arterial reconstruction.