Dual effect of heat shock on DNA replication and genome integrity

Heat shock (HS) is one of the better-studied exogenous stress factors. However, little is known about its effects on DNA integrity and the DNA replication process. In this study, we show that in G1 and G2 cells, HS induces a countable number of double-stranded breaks (DSBs) in the DNA that are marked by γH2AX. In contrast, in S-phase cells, HS does not induce DSBs but instead causes an arrest or deceleration of the progression of the replication forks in a temperature-dependent manner. This response also provoked phosphorylation of H2AX, which appeared at the sites of replication. Moreover, the phosphorylation of H2AX at or close to the replication fork rescued the fork from total collapse. Collectively our data suggest that in an asynchronous cell culture, HS might affect DNA integrity both directly and via arrest of replication fork progression and that the phosphorylation of H2AX has a protective effect on the arrested replication forks in addition to its known DNA damage signaling function.     

InvertNet: a new paradigm for digital access to invertebrate collections

InvertNet, one of the three Thematic Collection Networks (TCNs) funded in the first round of the U.S. National Science Foundation's Advancing Digitization of Biological Collections (ADBC) program, is tasked with providing digital access to ~60 million specimens housed in 22 arthropod (primarily insect) collections at institutions distributed throughout the upper midwestern USA. The traditional workflow for insect collection digitization involves manually keying information from specimen labels into a database and attaching a unique identifier label to each specimen. This remains the dominant paradigm, despite some recent attempts to automate various steps in the process using more advanced technologies. InvertNet aims to develop improved semi-automated, high-throughput workflows for digitizing and providing access to invertebrate collections that balance the need for speed and cost-effectiveness with long-term preservation of specimens and accuracy of data capture. The proposed workflows build on recent methods for digitizing and providing access to high-quality images of multiple specimens (e.g., entire drawers of pinned insects) simultaneously. Limitations of previous approaches are discussed and possible solutions are proposed that incorporate advanced imaging and 3-D reconstruction technologies. InvertNet couples efficient digitization workflows with a highly robust network infrastructure capable of managing massive amounts of image data and related metadata and delivering high-quality images, including interactive 3-D reconstructions in real time via the Internet.     

Eco-label conveys reliable information on fish stock health to seafood consumers

Concerns over fishing impacts on marine populations and ecosystems have intensified the need to improve ocean management. One increasingly popular market-based instrument for ecological stewardship is the use of certification and eco-labeling programs to highlight sustainable fisheries with low environmental impacts. The Marine Stewardship Council (MSC) is the most prominent of these programs. Despite widespread discussions about the rigor of the MSC standards, no comprehensive analysis of the performance of MSC-certified fish stocks has yet been conducted. We compared status and abundance trends of 45 certified stocks with those of 179 uncertified stocks, finding that 74% of certified fisheries were above biomass levels that would produce maximum sustainable yield, compared with only 44% of uncertified fisheries. On average, the biomass of certified stocks increased by 46% over the past 10 years, whereas uncertified fisheries increased by just 9%. As part of the MSC process, fisheries initially go through a confidential pre-assessment process. When certified fisheries are compared with those that decline to pursue full certification after pre-assessment, certified stocks had much lower mean exploitation rates (67% of the rate producing maximum sustainable yield vs. 92% for those declining to pursue certification), allowing for more sustainable harvesting and in many cases biomass rebuilding. From a consumer's point of view this means that MSC-certified seafood is 3-5 times less likely to be subject to harmful fishing than uncertified seafood. Thus, MSC-certification accurately identifies healthy fish stocks and conveys reliable information on stock status to seafood consumers.     

Xanthurenic Acid Binds to Neuronal G-Protein-Coupled Receptors That Secondarily Activate Cationic Channels in the Cell Line NCB-20

Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca²⁺ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.     

Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkA^NGFR) and/or the pan-neurotrophin glycoprotein receptor (p75^NTR). Therefore, a conjunctival myoFB model (TGFβ1-induced myoFB) was developed and characterized for cell viability/proliferation as well as αSMA, p75^NTR and trkA^NGFR expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75^NTR/trkA^NGFR, αSMA/TGFβ1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75^NTR, associated with a deregulation of both αSMA/TGFβ1 genes. Acute and chronic NGF exposures induced apoptosis in p75^NTR expressing myoFB, an effect counteracted by the specific trkA^NGFR and/or p75^NTR inhibitors. Focused single p75^NTR and double trkA^NGFR/p75^NTR knocking-down experiments highlighted the role of p75^NTR in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75^NTR. The trkA^NGFR/p75^NTR ratio in favor of p75^NTR characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.     

Bivalirudin in combination with heparin to control mesenchymal cell procoagulant activity

Islet and hepatocyte transplantation are associated with tissue factor-dependent activation of coagulation which elicits instant blood mediated inflammatory reaction, thereby contributing to a low rate of engraftment. The aim of this study was i) to evaluate the procoagulant activity of human adult liver-derived mesenchymal progenitor cells (hALPCs), ii) to compare it to other mesenchymal cells of extra-hepatic (bone marrow mesenchymal stem cells and skin fibroblasts) or liver origin (liver myofibroblasts), and iii) to determine the ways this activity could be modulated. Using a whole blood coagulation test (thromboelastometry), we demonstrated that all analyzed cell types exhibit procoagulant activity. The hALPCs pronounced procoagulant activity was associated with an increased tissue factor and a decreased tissue factor pathway inhibitor expression as compared with hepatocytes. At therapeutic doses, the procoagulant effect of hALPCs was inhibited by neither antithrombin activators nor direct factor Xa inhibitor or direct thrombin inhibitors individually. However, concomitant administration of an antithrombin activator or direct factor Xa inhibitor and direct thrombin inhibitor proved to be a particularly effective combination for controlling the procoagulant effects of hALPCs both in vitro and in vivo. The results suggest that this dual antithrombotic therapy should also improve the efficacy of cell transplantation in humans.     

Readmissions and Death after ICU Discharge: Development and Validation of Two Predictive Models

Introduction

Early discharge from the ICU is desirable because it shortens time in the ICU and reduces care costs, but can also increase the likelihood of ICU readmission and post-discharge unanticipated death if patients are discharged before they are stable. We postulated that, using eICU® Research Institute (eRI) data from >400 ICUs, we could develop robust models predictive of post-discharge death and readmission that may be incorporated into future clinical information systems (CIS) to assist ICU discharge planning.

Methods

Retrospective, multi-center, exploratory cohort study of ICU survivors within the eRI database between 1/1/2007 and 3/31/2011. Exclusion criteria: DNR or care limitations at ICU discharge and discharge to location external to hospital. Patients were randomized (2∶1) to development and validation cohorts. Multivariable logistic regression was performed on a broad range of variables including: patient demographics, ICU admission diagnosis, admission severity of illness, laboratory values and physiologic variables present during the last 24 hours of the ICU stay. Multiple imputation was used to address missing data. The primary outcomes were the area under the receiver operator characteristic curves (auROC) in the validation cohorts for the models predicting readmission and death within 48 hours of ICU discharge.

Results

469,976 and 234,987 patients representing 219 hospitals were in the development and validation cohorts. Early ICU readmission and death was experienced by 2.54% and 0.92% of all patients, respectively. The relationship between predictors and outcomes (death vs readmission) differed, justifying the need for separate models. The models for early readmission and death produced auROCs of 0.71 and 0.92, respectively. Both models calibrated well across risk groups.

Conclusions

Our models for death and readmission after ICU discharge showed good to excellent discrimination and good calibration. Although prospective validation is warranted, we speculate that these models may have value in assisting clinicians with ICU discharge planning.     

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+) -transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism.