Laparoscopical intrauterine insemination with different doses of fresh,conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.     

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.     

Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets

Low oxygen (O(2)) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O(2)). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.     

The hyporheic zone as a source of dissolved organic carbon and carbon gases to a temperate forested stream

The objective of this study was to examine chemical changes in porewaters that occur over small scales (cm) as groundwater flows through the hyporheic zone and discharges to a stream in a temperate forest of northern Wisconsin. Hyporheic-zone porewaters were sampled at discrete depths of 2, 10, 15, 61, and 183 cm at three study sites in the study basin. Chemical profiles of dissolved organic carbon (DOC), CO₂, CH₄, and pH show dramatic changes between 61 cm sediment depth and the water-sediment interface. Unless discrete samples at small depth intervals are taken, these chemical profiles are not accounted for. Similar trends were observed at the three study locations, despite each site having very different hydraulic-flow regimes. Increases in DOC concentration by an order of magnitude from 61 to 15 cm depth with a corresponding decrease in pH and rapid decreases in the molecular weight of the DOC suggest that aliphatic compounds (likely organic acids) are being generated in the hyporheic zone. Estimated efflux rates of DOC, CO₂, and CH₄ to the stream are 6.2, 0.79, 0.13 moles m² d^-1, respectively, with the vast majority of these materials produced in the hyporheic zone. Very little of these materials are accounted for by sampling stream water, suggesting rapid uptake and/or volatilization.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

Governance options for science–policy interfaces on biodiversity and ecosystem services: comparing a network versus a platform approach

Science–policy-interfaces (SPIs) are expected to go beyond the linear model of scientific policy advice through creating spaces for exchange and dialogue between ‘policy’ and ‘knowledge’. Given that most environmental issues require inter- and transdisciplinary approaches, SPIs must take into account a variety of knowledge types, views and interests of scientists, policymakers and other decision makers. Moreover, acceptance and durability of SPIs depend largely on their perceived legitimacy and the credibility of their knowledge-gathering processes, providing additional challenges for their internal organisation. As the interplay between different knowledge types and decision making is far from neutral, a reflexive approach is required in the design of an SPI so that it is capable of learning from past experiences. The aim of this article is to discuss which governance arrangements could best support the development of an effective and legitimate SPI for European biodiversity politics. We analyse different options for facilitating the implementation of a ‘Network of Knowledge’ approach. This approach has been developed to improve the interface between diverse knowledge-holder communities and decision making processes for biodiversity and ecosystem services—a field where multi-scalar and multi-dimensional problems arise. In this article, we develop and discuss two stylized extreme governance models as our starting point: an `informal network model´, which almost entirely depends on the dedication of individuals, versus a more formalized `platform model´, predominantly based on the needs and interests of the organisations involved. We discuss the pros and cons of each of these models in reaching their objectives and in developing sound governing processes for a ‘Network of Knowledge’. From this discussion, we derive a recommended design for the reflexive governance of such a network in the context of the European Union and finish by discussing some more general lessons learnt.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

The Patient- And Nutrition-Derived Outcome Risk Assessment Score (PANDORA): Development of a Simple Predictive Risk Score for 30-Day In-Hospital Mortality Based on Demographics,Clinical Observation,and Nutrition

ObjectiveTo develop a simple scoring system to predict 30 day in-hospital mortality of in-patients excluding those from intensive care units based on easily obtainable demographic, disease and nutrition related patient data.MethodsScore development with general estimation equation methodology and model selection by P-value thresholding based on a cross-sectional sample of 52 risk indicators with 123 item classes collected with questionnaires and stored in an multilingual online database.SettingWorldwide prospective cross-sectional cohort with 30 day in-hospital mortality from the nutritionDay 2006-2009 and an external validation sample from 2012.ResultsWe included 43894 patients from 2480 units in 32 countries. 1631(3.72%) patients died within 30 days in hospital. The Patient- And Nutrition-Derived Outcome Risk Assessment (PANDORA) score predicts 30-day hospital mortality based on 7 indicators with 31 item classes on a scale from 0 to 75 points. The indicators are age (0 to 17 points), nutrient intake on nutritionDay (0 to 12 points), mobility (0 to 11 points), fluid status (0 to 10 points), BMI (0 to 9 points), cancer (9 points) and main patient group (0 to 7 points). An appropriate model fit has been achieved. The area under the receiver operating characteristic curve for mortality prediction was 0.82 in the development sample and 0.79 in the external validation sample.ConclusionsThe PANDORA score is a simple, robust scoring system for a general population of hospitalised patients to be used for risk stratification and benchmarking.