bluuebunny/crossref_metadata_2025 huggingface dataset:
232 GENERATION OF TRANSGENIC PIGS CARRYING A SMALL INTERFERING RNA VECTOR DIRECTED AGAINST TISSUE FACTOR EXPRESSION
Authors: Ahrens H. E. , Petersen B. , Queisser A. L. , Herrmann D. , Kues W. , Holler S. , Hassel P. , Lemme E. , Lucas-Hahn A. , Niemann H.
Published: December 2011

The acute vascular rejection (AVR) remains the main hurdle for long-term survival of a porcine xenograft after transplantation into primates. Immunological reactions and molecular incompatibilities can lead to endothelial activation and microvascular thrombosis. Knock-down of tissue factor (TF) could be a promising mechanism to prevent AVR. Tissue factor is a key molecule of the extrinsic coagulation pathway. It functions as cell surface receptor for coagulation factor VIIa and thereby initiates thrombin formation. Since TF knockout was lethal in the mouse model (Toomey et al. 1996 Blood 88, 1583–1587), we tested different small interfering (si) RNAs in their efficiency to knock down TF in a porcine cell line. Two siRNAs reduced the mRNA level of TF to <3% compared with wild type controls as determined by RT-PCR. Subsequently, porcine fetal fibroblasts (PFF) were co-transfected with constructs coding for one of the most promising siRNAs and a DsRed vector, which confers neomycin resistance and red fluorescence. Transfected cells were selected with neomycin for 14 days and checked for the occurrence of fluorescence. Integration of the siRNA vector was confirmed by PCR. Cell clones positive for TF knock-down siRNA served as donor cells for somatic cell nuclear transfer (SCNT). Successfully reconstructed embryos were transferred to synchronised recipient sows. Eight transfers resulted in 4 pregnancies. One foster animal was sacrificed on Day 35 of gestation. All of the 3 resulting fetuses had integrated the TF knock-down siRNA as shown by PCR and will be used for recloning. Additionally, we plan to determine vector integration by Southern blot analysis and siRNA and TF expression level by real time-PCR. The remaining 3 sows will be allowed to carry to full term for further investigations. The functionality of the knock-down will be tested in a tumo necrosis factor-α induced assay using cultured endothelial cells of knock-down piglets and of wild type counterparts because exposure to inflammation mediators stimulates expression of TF and adhesion molecules in the endothelium. Our genetically modified TF transgenic pigs can be combined with already existing genetic modifications to generate multi-transgenic pigs. This model would be of great importance to prolong xenograft survival and move porcine xenografts to the preclinical level.
This project was funded by the EU project ‘Xenome’ (LSHB-CT-2006-037377).
233 GENDER-UNSPECIFIC KNOCKOUT OF THE GGTA1 GENE IN PIGS USING ZINC FINGER NUCLEASES
Authors: Hauschild J. , Petersen B. , Santiago Y. , Queisser A. L. , Carnwath J. W. , Lucas-Hahn A. , Zhang L. , Meng X. , Gregory P. D. , Schwinzer R. , Cost G. J. , Niemann H.
Published: December 2011

A knockout (KO) of the porcine α1,3-galactosyltransferase (GGTA1) gene is crucial for controlling the hyperacute rejection after pig-to-human xenotransplantation. Porcine kidney and cardiac xenografts from Gal-KO pigs showed prolonged survival after transplantation into baboons. Unfortunately, knockouts produced by conventional targeting (homologous recombination) are rare events and normally do not lead to biallelic KO. Zinc-finger nucleases (ZFN) have been shown to be much more efficient by inducing mutations via specific cleavage followed by nonhomologous end joining (NHEJ). Zinc-finger nucleases do not require antibiotic selection. Here, we used designed ZFN to specifically target exon 9 of the GGTA1 gene encoding the catalytic domain of the Gal-transferase. Recently, we generated female pigs with a GGTA1-KO using ZFN (Hauschild et al. 2011 PNAS 108, 12 013–12 017). Here, we investigated whether cells of a male cell line are susceptible to ZFN-mediated genome editing in a comparable manner. Male porcine fetal fibroblasts (3 × 106) were co-transfected with a ZFN-plasmid pair (7.5 μg each) by electroporation at 250 V and 400 μF. One week after transfection, a Cel-I assay revealed a NHEJ rate of 5.7% of all alleles in the cell population. After magnetic bead selection, Gal-expression was analysed by fluorescence-activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. Ninety-five percent of the cells were free of Gal epitopes, indicating a biallelic KO. These Gal-negative cells served as donor cells in somatic cell nuclear transfer (SCNT). In total, 507 transgenic embryos were transferred into 6 recipient sows. By obtaining live animals by SCNT after transfer of male ZFN-GGTA1-KO embryos, we will have produced female and male ZFN-KO pigs, which can be used for further breeding experiments to circumvent the extensive and relative inefficient recloning method. These results show that ZFN work independent of the sex of the cells and that a biallelic Gal-KO can be produced in male cells by using the ZFN technology. This technology could benefit both agriculture and biomedicine and establishes the pig as a model for human diseases.
THE USE OF RUMINANTS FOR BIOMEDICAL RESEARCH IN PERINATALOGY
Authors: Chavatte-Palmer Pascale , Tarrade Anne , Tsatsaris Vassilis , Morel Olivier
Published: December 2011

As a consequence of obvious ethical considerations concerning what is feasible or not in the field of human biomedical research, animal models are of critical importance both for human and veterinary medicine and cognitive scientists. Many species have been and are currently used as animal models. Rodents and lagomorphs are the most popular, essentially because of their low cost, handling and rearing facilities, limited ethical impact, and the availability of a wide range of genetic research tools in these species. Nevertheless, these models present some limitations. The physiological mechanisms observed in these species might be far from those of humans. For example, due to the large number of embryos and short gestation length, rodents and lagomorphs are not very suitable in several fields of perinatal research. Large animal species are required when surgical approaches or new medical devices have to be evaluated. The pig is widely used in these situations, as well as ruminants such as the sheep and the goat. Concerning physiological, anatomical and genetic considerations, large primates could be considered as the “gold standard” animal model because of their important similarities with humans. However, their use for biomedical research is greatly limited by their behavioral and social organization, raising important ethical questions, and their elevated cost. A very large number of experiments using ruminants as animal models have been published. A rapid bibliometric analysis performed using the Pubmed database from 1969 to 2010 retrieved 1108 literature reviews using the Mesh keywords “ruminant & animal model”. Six hundred and twelve references were available only for the year 2009 using the same Mesh research and covering all types of publications. The sheep is the most widely used ruminant model, and no less than 5393 publications can be found using the Mesh keywords “sheep & animal model”. The current presentation will focus on the most outstanding examples of great biomedical advances carried out with ruminant as models in the field of perinatal research. In this paper, the pivotal role of ruminants in the evaluation and development of new pharmacological treatments, new invasive procedures in fetuses, gestational imaging and genetic approaches will be developed, with their clinical context, including our own work on feto-placental imaging and the assessment of placental blood flow using quantitative 3D Doppler.
THE IMPORTANCE OF LARGE ANIMAL MODELS FOR TRANSLATIONAL RESEARCH IN BONE TISSUE ENGINEERING
Authors: Hollister S. J. , Wheeler M. B. , Feinberg S. E. , Murphy W. L.
Published: December 2011

The translation of bone tissue engineering (BTE) research to clinical use has been absymal1. Outside of bone void filler biomaterials, only Bone Morphogenetic Protein 2 (BMP2) has made significant inroads to clinical practice, and even BMP2 use has been associated with significant complications including death, dysphagia, and ectopic bone formation. The dearth of BTE products can be attributed to two main causes: (1) the need to develop BTE systems, that successfully integrate scaffolds, growth factors like BMP2 and cells and (2) the need to adapt and implement such systems for a wide variety of clinical indications in CranioMaxilloFacial (CMF), Spine and Orthopedic Surgery. Of course, to fully develop BTE systems (Issue 1) and adapt them to realistic clinical indications, we must be able to test such systems in bone defects that are as close to the human situation as possible. Thus, the use of domestic large animals for bone tissue engineering is critical, as these animals provide challenges in both defect volume and functional loading that can mimic the human situation. In addition, FDA approval for BTE products either through a 510K or IDE/IND/PMA pathway requires the use of a large pre-clinical animal model. However, despite this need, only approximately 60 large animal bone tissue-engineering studies have been published in the past 10 years. Furthermore, NIH has funded only 8% of these studies, and of the 17 bone tissue engineering studies supported by NIH in 2010, only three utilized a large animal model, and none of these used an animal larger than a rabbit. Clearly, increased translation and regulatory approval of BTE therapies will require greater testing in large animal models. We will discuss the current dearth of relevant pre-clinical studies in BTE, and present our work addressing these issues by developing BTE systems (integrated scaffold, growth factor and stem-cell constructs) and testing these systems for realistic clinical applications using the Yorkshire and other swine species as a large pre-clinical animal model. We will detail our work in developing BTE systems for CMF reconstruction and spine fusion in the swine model.

Reference
Hollister S. J. and Murphy W. L. Scaffold translation: barriers between concept and clinic. Tissue Eng. B. (in press).
4 EFFECT OF A PROLONGED AROMATASE INHIBITOR TREATMENT ON PRE-OVULATORY OVARIAN FOLLICLES IN CATTLE
Authors: Yapura J. , Singh J. , Mapletoft R. J. , Pierson R. , Rogan D. , Adams G. P.
Published: December 2011

Letrozole, a non-steroidal aromatase inhibitor, prevents the body from producing its own oestrogen. The potential use of this compound for herd synchronization is supported by previous studies in which letrozole treatment increased mean plasma LH concentrations, prolonged the period of dominance of the extant dominant follicle and delayed emergence of the next follicular wave. Heifers given a 3-day regimen of letrozole exhibited greater corpus luteum diameter indicative of a luteotrophic effect. The objective of the present study was to test the hypothesis that letrozole treatment during the development of the preovulatory follicular wave will delay ovulation. Post-pubertal beef heifers were given 2 luteolytic doses of PGF (12 h apart) and monitored by ultrasonography for ovulation. Ovarian follicular wave emergence was synchronized by ultrasound-guided transvaginal follicular ablation 5 to 8 days after PGF-induced ovulation (Day –1 = follicular ablation, Day 0 = wave emergence) and a luteolytic dose of PGF was given 60 and 72 h later. On Day 1, heifers were divided randomly into 2 groups (n = 15/group) and given an intravaginal device containing 1 g of letrozole or a blank device (control). The intravaginal devices were removed on Day 7, or at the time of ovulation, whichever occurred first. The ovaries were monitored by ultrasonography and a blood sample was collected daily from day of ablation to 12 days post-ovulation. Single point measurements were analysed by t-tests and serial data were analysed by analysis of variance for repeated measures. Multiple contrasts were made by Tukey's test. The interval from placement of the intravaginal device to ovulation was longer in letrozole-treated animals (6.1 ± 0.25 vs 5.1 ± 0.26 days, P < 0.01). Compared with controls, the day-to-day diameter profile of the dominant follicle of the ovulatory wave was larger (P < 0.05) and the maximum diameter greater (14.6 ± 0.51 vs 12.4 ± 0.53 mm; P < 0.01) in letrozole-treated heifers. The diameter profile of the corpus luteum formed post-letrozole treatment did not differ between groups; however, plasma P4 concentrations were higher (P < 0.01) in heifers treated with letrozole. In summary, a slow-release intravaginal letrozole device delayed ovulation by 24 h and induced the formation of a corpus luteum that secreted higher levels of progesterone. A slow-release intravaginal letrozole device may become useful for the development of an aromatase inhibitor-based protocol to control ovulation for herd synchronization and to enhance fertility by increasing circulating progesterone concentrations during the first 7 days post-AI or embryo transfer in cattle.
Supported by the Natural Sciences and Engineering Research Council of Canada and Bioniche Life Sciences Inc.
57 DIETARY CLA SUPPLEMENTATION AFFECTS LUTEAL GENE EXPRESSION AND PERIPHERAL BLOOD PROGESTERONE CONCENTRATION IN CATTLE
Authors: Stinshoff H. , Onnen-Lübben E. , Wilkening S. , Hanstedt A. , Bollwein H. , Wrenzycki C.
Published: December 2011

Shortly after parturition the metabolic situation of high-yielding dairy cows is often dominated by a negative energy balance. These effects affect the whole animal and may especially be detected in the reproductive tract, where they result in reduced fertility. An oral supplementation with dietary fats is often used to counteract by reducing milk fat content and, thus, supplying the individual animal with an increased amount of energy. The focus of the present study was to analyse the effects of an oral supplementation with conjugated linoleic acids (CLA) on corpus luteum (CL) function. Healthy Holstein-Friesian cows and heifers were randomly allocated to 2 treatment groups (Group 1: 50 g of CLA day–1 per animal, 2 heifers, 6 cows; Group 2: 100 g of CLA day–1 per animal, 2 heifers, 6 cows) and 1 control group (Ctl; 0 g of CLA day–1 per animal, 3 heifers, 4 cows). Feeding of the supplement began shortly after calving. After calving, all animals were subjected to a standard synchronisation protocol and experienced AI on Day 59 ± 3. Following AI, transvaginal biopsies of the corpus luteum were obtained of pregnant (Group I: n = 4; Group II: n = 4; Ctl: n = 4) and nonpregnant (Group I: n = 4; Group II: n = 4; Ctl: n = 3) animals on Days 6, 13 and 20 post-AI. Animals deemed pregnant on Day 28 were again biopsied on Day 42. Additionally, blood samples were taken from the vena sacralis mediana at the time of each biopsy. The biopsies were analysed regarding the relative abundance of 8 gene transcripts (VEGF, ECE1, PLA2G4A, PTGS2, PTGFR, PPARG, STAR and HSD3B1) via RT-qPCR. Blood samples were analysed for their concentration of progesterone through a radioimmunoassay (RIA). Statistical analysis for both datasets was performed via a 3-way ANOVA with adjoining Tukey test. The expression of 7 of these genes was affected by 1, 2, or all 3 of the following factors: day of cycle (VEGF, ECE1, PLA2G4A, PTGFR, STAR and HSD3B1), pregnancy status (ECE1, PTGFR and HSD3B1) and CLA supplementation (ECE1, PTGS2, PTGFR, STAR and HSD3B1). The effects of the CLA supplementation could be seen as a down-regulation in the mentioned gene transcripts. Progesterone concentrations differed significantly in dependency of the pregnancy status (significantly higher in pregnant vs nonpregnant individuals) of the animals, as well as during the days of the oestrous cycle (physiological progesterone curve with highest values on Day 13 of these samples). An effect of the oral supplementation with CLA could be detected during the early luteal phase (Day 6) where animals that had received 100 g of CLA day–1 had a significantly lower blood progesterone concentration than those receiving 50 g of CLA day–1 or no CLA. In conclusion, dietary CLA supplementation has an effect on luteal gene expression and functionality.
The authors thank the DFG (German Research Foundation) for their financial support (PAK286/1; WR154/1-1).
59 HISTONE DEACETYLASE 1 KNOCK-DOWN IN BOVINE FIBROBLAST CELLS AND CLONED EMBRYOS
Authors: Wang Z. W. , Zhang P. , Zhang S. , Ma X. , Yin Y. P. , Tang B. , Li Z. Y.
Published: December 2011

Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells. It was thought to be the most important enzyme in the regulation of histone deacetylation process. However, the function of HDAC1 in bovine fibroblast cells and nuclear transfer embryos is not clear. In the present study, sh299 (5′GCAAGCAGATGCAGAGATTTCAAGA GAATCTCTGCATCTGCTTGCTT3′) targeting of HDAC1 mRNA sequence was designed in the PGP/U6/GFP vector (short hairpin RNA, shRNA, expression vector). The sh299 vector was transfected into bovine fibroblast cells by transfection reagent FuGENE HD and the positive cells were identified by the expression of green fluorescent protein (GFP). Histone deacetylase 1 down-regulation in bovine fibroblast cells was measured by quantitative real-time PCR (qRT-PCR with the 2–ΔΔCT method) at 48 h after sh299 vector transfection at mRNA level. Immunocytochemistry was performed at 96 h after sh299 vector transfection to examine the HDAC1 protein level. Bovine fibroblast cells of the control group, negative control vector transfection group and sh299 vector transfection group were used as donor cells for nuclear transfer. Cleavage rates and expression of HDAC1 mRNA in bovine cloned embryos were examined at 48 h after nuclear transfer. Blastocyst rates and total cell numbers of blastocysts were examined on Day 7 post-nuclear transfer. Data were analysed with Statistics Production for Service Solution (version 16.0; SPSS) by 1-way ANOVA. A value of P < 0.05 was considered to be significantly different. Our results showed that the expression level of HDAC1 was significantly reduced by transfection of the sh299 expression vector. The GFP-positive cells showed decreased signal for HDAC1 by immunocytochemistry. It was suggested that the transfection of the sh299 expression vector reduced HDAC1 expression in bovine fibroblast cells at both mRNA level and protein level. Following nuclear transfer, expression of HDAC1 was significantly reduced in the sh299 vector transfection group (donor cells were transfected by the sh299 vector) compared to the other 2 groups. No significant difference (P > 0.05) was seen in cleavage rates among bovine cloned embryos in the sh299 vector transfection group (52.3 ± 3.4%), control group (51.1 ± 5.4%) and negative control vector transfection group (56.2 ± 3.1%). However, blastocyst rates and total cell numbers of blastocysts were significantly lower (P < 0.05) in the sh299 vector transfection group (4.2 ± 1.3% and 75.4 ± 9.2, n = 89) compared to the control group (18.2 ± 3.7% and 97.6 ± 7.3, n = 78) and negative control vector transfection group (18.9 ± 1.7% and 104.2 ± 10.3, n = 83). In conclusion, HDAC1 down-regulation in bovine fibroblast cells and cloned embryos by the sh299 expression vector indicated that HDAC1 was essential for the development of bovine cloned embryos.
This work was supported by the grant from National Transgenic Animal Program (No.2009ZX08007-004B) in China.
Economic and energetic analysis of capturing CO
2
from ambient air
Authors: House Kurt Zenz , Baclig Antonio C. , Ranjan Manya , van Nierop Ernst A. , Wilcox Jennifer , Herzog Howard J.
Published: December 2011

Capturing carbon dioxide from the atmosphere (“air capture”) in an industrial process has been proposed as an option for stabilizing global CO
2
concentrations. Published analyses suggest these air capture systems may cost a few hundred dollars per tonne of CO
2
, making it cost competitive with mainstream CO
2
mitigation options like renewable energy, nuclear power, and carbon dioxide capture and storage from large CO
2
emitting point sources. We investigate the thermodynamic efficiencies of commercial separation systems as well as trace gas removal systems to better understand and constrain the energy requirements and costs of these air capture systems. Our empirical analyses of operating commercial processes suggest that the energetic and financial costs of capturing CO
2
from the air are likely to have been underestimated. Specifically, our analysis of existing gas separation systems suggests that, unless air capture significantly outperforms these systems, it is likely to require more than 400 kJ of work per mole of CO
2
, requiring it to be powered by CO
2
-neutral power sources in order to be CO
2
negative. We estimate that total system costs of an air capture system will be on the order of $1,000 per tonne of CO
2
, based on experience with as-built large-scale trace gas removal systems.
Notch1 regulates the expression of the multidrug resistance gene
ABCC1
/
MRP1
in cultured cancer cells
Authors: Cho Sungpil , Lu Meiling , He Xiaolong , Ee Pui-Lai Rachel , Bhat Uppoor , Schneider Erasmus , Miele Lucio , Beck William T.
Published: December 2011

Multidrug resistance (MDR) is a barrier to successful cancer chemotherapy. Although MDR is associated with overexpression of ATP-binding cassette (ABC) membrane transporters, mechanisms behind their up-regulation are not entirely understood. The cleaved form of the Notch1 protein, intracellular Notch1 (N1
IC
), is involved in transcriptional regulation of genes. To test whether Notch1 is involved in the expression of multidrug resistance-associated protein 1 (ABCC1/MRP1; herein referred to as ABCC1), we measured N1
IC
and presenilin 1 (PSEN1), the catalytic subunit of γ-secretase required for Notch activation. We observed higher levels of N1
IC
and PSEN1 proteins as well as higher activity of N1
IC
in ABCC1-expressing MDR MCF7/VP cells compared with parental MCF7/WT cells. Reducing N1
IC
levels in MCF7/VP cells with either a γ-secretase inhibitor or shRNA led to reduction of ABCC1. By contrast, ectopic expression of N1
IC
in MCF7/WT cells led to increased expression of ABCC1 and associated drug resistance, consistent with expression of this transporter. Inhibition of ABCC1 reversed drug resistance of N1
IC
-overexpressing stable cells. Using an ABCC1 promoter construct, we observed both its reduced transcriptional activity after blocking the generation of N1
IC
and its increased transcriptional activity in stable cells overexpressing N1
IC
. ChIP and gel-shift assays revealed an interaction between a specific promoter region of ABCC1 and the N1
IC
-activated transcription factor CBF1, suggesting that the regulation of ABCC1 expression by Notch1 is mediated by CBF1. Indeed, deletion or site-directed mutagenesis of these CBF1 binding sites within the ABCC1 promoter region attenuated promoter-reporter activity. Overall, our results reveal a unique regulatory mechanism of ABCC1 expression.
Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach
Authors: Braun Nathalie , Zacharias Martin , Peschek Jirka , Kastenmüller Andreas , Zou Juan , Hanzlik Marianne , Haslbeck Martin , Rappsilber Juri , Buchner Johannes , Weinkauf Sevil
Published: December 2011

The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how “heterogeneity” is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.
Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses
Authors: Sandbulte Matthew R. , Westgeest Kim B. , Gao Jin , Xu Xiyan , Klimov Alexander I. , Russell Colin A. , Burke David F. , Smith Derek J. , Fouchier Ron A. M. , Eichelberger Maryna C.
Published: December 2011

Seasonal epidemics caused by influenza virus are driven by antigenic changes (drift) in viral surface glycoproteins that allow evasion from preexisting humoral immunity. Antigenic drift is a feature of not only the hemagglutinin (HA), but also of neuraminidase (NA). We have evaluated the antigenic evolution of each protein in H1N1 and H3N2 viruses used in vaccine formulations during the last 15 y by analysis of HA and NA inhibition titers and antigenic cartography. As previously shown for HA, genetic changes in NA did not always lead to an antigenic change. The noncontinuous pattern of NA drift did not correspond closely with HA drift in either subtype. Although NA drift was demonstrated using ferret sera, we show that these changes also impact recognition by NA-inhibiting antibodies in human sera. Remarkably, a single point mutation in the NA of A/Brisbane/59/2007 was primarily responsible for the lack of inhibition by polyclonal antibodies specific for earlier strains. These data underscore the importance of NA inhibition testing to define antigenic drift when there are sequence changes in NA.
Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1α
Authors: Berda-Haddad Yaël , Robert Stéphane , Salers Paul , Zekraoui Leila , Farnarier Catherine , Dinarello Charles A. , Dignat-George Françoise , Kaplanski Gilles
Published: December 2011

Sterile inflammation resulting from cell death is due to the release of cell contents normally inactive and sequestered within the cell; fragments of cell membranes from dying cells also contribute to sterile inflammation. Endothelial cells undergoing stress-induced apoptosis release membrane microparticles, which become vehicles for proinflammatory signals. Here, we show that stress-activated endothelial cells release two distinct populations of particles: One population consists of membrane microparticles (<1 μm, annexin V positive without DNA and no histones) and another larger (1–3 μm) apoptotic body-like particles containing nuclear fragments and histones, representing apoptotic bodies. Contrary to present concepts, endothelial microparticles do not contain IL-1α and do not induce neutrophilic chemokines in vitro. In contrast, the large apoptotic bodies contain the full-length IL-1α precursor and the processed mature form. In vitro, these apoptotic bodies induce monocyte chemotactic protein-1 and IL-8 chemokine secretion in an IL-1α–dependent but IL-1β–independent fashion. Injection of these apoptotic bodies into the peritoneal cavity of mice induces elevated serum neutrophil-inducing chemokines, which was prevented by cotreatment with the IL-1 receptor antagonist. Consistently, injection of these large apoptotic bodies into the peritoneal cavity induced a neutrophilic infiltration that was prevented by IL-1 blockade. Although apoptosis is ordinarily considered noninflammatory, these data demonstrate that nonphagocytosed endothelial apoptotic bodies are inflammatory, providing a vehicle for IL-1α and, therefore, constitute a unique mechanism for sterile inflammation.
Electrostatic origin of the mechanochemical rotary mechanism and the catalytic dwell of F1-ATPase
Authors: Mukherjee Shayantani , Warshel Arieh
Published: December 2011

Understanding the nature of energy transduction in life processes requires a quantitative description of the energetics of the conversion of ATP to ADP by ATPases. Previous attempts to do so have provided an interesting insight but could not account for the rotary mechanism by a nonphenomenological structure/energy description. In particular it has been very challenging to account for the observations of the 80° and 40° rotational substates, without any prior information about such states in the simulation procedure. Here we use a coarse-grained model of F1-ATPase and generate, without the adjustment of phenomenological parameters, a structure-based free energy landscape that reproduces the energetics of the mechanochemical process. It is found that the landscape along the relevant rotary path is determined by the electrostatic free energy and not by steric effects. Furthermore, the generated surface and the corresponding Langevin dynamics simulations identify a hidden conformational barrier that provides a new fundamental interpretation of the catalytic dwell and illuminate the nature of the energy conversion process.
Larval Surveys Indicate Low Levels of Endangered Pallid Sturgeon Reproduction in the Middle Mississippi River
Authors: Boley Ryan M. , Heist Edward J.
Published: December 2011

The pallid sturgeon Scaphirhynchus albus is an endangered riverine species that is less abundant than the sympatric shovelnose sturgeon S. platorynchus with which it hybridizes. The two species are morphologically similar, and due to morphological variation within species, allometry, and the occurrence of morphological intermediates, morphological identification of specimens can be problematic. When the pallid sturgeon was listed under the U.S. Endangered Species Act in 1990, perceived threats included habitat loss due to habitat alteration (dams and channelization), hybridization, and commercial harvest. Previous studies estimated the ratio of adult pallid sturgeon to adult shovelnose sturgeon to be approximately 1:77 in the middle Mississippi River (MMR; the reach between the Missouri River confluence and the Ohio River confluence). One study using morphological identification estimated that 8–26% of Scaphirhynchus larvae collected in the MMR during May and June were pallid sturgeon. Given that larval pallid sturgeon, shovelnose sturgeon, and their hybrids have overlapping morphologies, DNA markers may provide more accurate species identification. The present study aimed to estimate relative larval abundances of pallid sturgeon, shovelnose sturgeon, and hybrids in the MMR. We genotyped 583 Scaphirhynchus larvae collected from the MMR at 16 DNA microsatellite loci and determined that 581 larvae were shovelnose sturgeon, one was a hybrid, and one was a pallid sturgeon. This study is the first to provide genetically verified larval abundance estimates of Scaphirhynchus species in the MMR. The present data indicate that the rate of pallid sturgeon reproduction relative to shovelnose sturgeon reproduction is an order of magnitude lower than expectations based on adult ratios and that hybrid sturgeon larvae are as common as pure pallid sturgeon.
A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation
Authors: Westerman Bart A. , Braat A. Koen , Taub Nicole , Potman Marko , Vissers Joseph H.A. , Blom Marleen , Verhoeven Els , Stoop Hans , Gillis Ad , Velds Arno , Nijkamp Wouter , Beijersbergen Roderick , Huber Lukas A. , Looijenga Leendert H.J. , van Lohuizen Maarten
Published: December 2011

Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. We used a genome-wide short hairpin RNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3 or Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation, and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4-driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were overexpressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation toward pluripotency and up-regulated the pluripotency-related genes Esrrb, Rex1, Tcl1, and Sox2. We also found that human germ cell tumors (GCTs) express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the noninvasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive GCT cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells and in germ cell cancer.
CEACAM1 dampens antitumor immunity by down-regulating NKG2D ligand expression on tumor cells
Authors: Chen Zhangguo , Chen Lanfen , Baker Kristi , Olszak Torsten , Zeissig Sebastian , Huang Yu-Hwa , Kuo Timothy T. , Mandelboim Ofer , Beauchemin Nicole , Lanier Lewis L. , Blumberg Richard S.
Published: December 2011

Although carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CEACAM1) has been viewed as a tumor suppressor, increasing clinical evidence shows that high levels of CEACAM1 expression on tumors correlates with poor prognosis and high risk of metastasis. Here, we examined the consequences of CEACAM1 expression on tumor cells. We show that tumor cell–associated CEACAM1 causes intracellular retention of various NKG2D ligands in mouse and human tumor cells. CEACAM1-silenced tumor cells expressed more cell surface NKG2D ligands and exhibited greater sensitivity to natural killer cell–mediated cytolysis in vitro and rejection in vivo. Our studies reveal a novel mechanism through which CEACAM1-bearing tumor cells may escape immune-surveillance.
Localization of Cell Wall Polysaccharides in Normal and Compression Wood of Radiata Pine: Relationships with Lignification and Microfibril Orientation
Authors: Donaldson Lloyd A. , Knox J. Paul
Published: December 2011

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.
Adolescents with congenital adrenal hyperplasia because of 21‐hydroxylase deficiency have vascular dysfunction
Authors: Harrington Jennifer , Peña Alexia S. , Gent Roger , Hirte Craig , Couper Jennifer
Published: December 2011

Context  Patients with congenital adrenal hyperplasia (CAH) because of 21‐hydroxylase deficiency have multiple vascular risk factors. Young adults with CAH have increased intima media thickness, but there have been no studies of vascular function and structure in children with CAH.

Objective  To establish whether children with CAH have reduced vascular function and increased carotid intima media thickness (cIMT) when compared to healthy and obese children.

Design and Patients  Cross‐sectional study of 14 patients (14·8 years ± 3·2, seven boys) with CAH secondary to 21‐hydroxylase deficiency compared to 28 obese and 53 healthy controls.

Measurements  All subjects had assessment of endothelial function flow‐mediated dilatation, (FMD), smooth muscle function glyceryl tri‐nitrate dilatation (GTN) and cIMT. Anthropometric data, resting blood pressure and biochemical variables were also measured.

Results  Congenital adrenal hyperplasia subjects had significantly reduced FMD (4·5 ± 3·0%vs 7·5 ± 5·2%; P = 0·04) and GTN (17·2 ± 1·6%vs 28·4 ± 8·4%; P < 0·001) when compared to controls and the impairment was comparable to the obese cohort. There was no significant difference in cIMT between groups. CAH subjects had increased homoeostasis model of assessment‐insulin resistance [HOMA‐IR 2·5 (0·2–2·9) vs 1·8 (0·5–4·2); P = 0·04], waist‐to‐height ratio (0·47 ± 0·05 vs 0·44 ± 0·04; P = 0·02) and higher systolic blood pressure Z score (0·29 ± 0·9 vs−0·24 ± 0·64, P = 0·01) compared to healthy controls but not when compared to obese controls.

Conclusions  Subjects with CAH have evidence of vascular dysfunction by adolescence.
Portuguese bee pollen: palynological study, nutritional and microbiological evaluation
Authors: Estevinho Leticia M. , Rodrigues Sandra , Pereira Ana P. , Feás Xesús
Published: December 2011

Bee pollen is one of the richest and purest natural foods, used in the folk medicine, clinical practices, and food and pharmaceutical industries. This work aims to characterise, for the first time in Portugal, the palynological origin, nutritional value and microbiological safety of this beehive product. It identified ten botanical families, and the more frequent were Boraginaceae and Ericaceae. We have evaluated the water content, aw, pH, reducing sugars, ash, carbohydrate, proteins, lipids, fatty acids and energy. The microorganisms studied were the aerobic mesophiles, moulds and yeasts, faecal coliforms, Escherichia coli, Staphylococcus aureus, Salmonella and sulphite‐reducing clostridia. The studied pollens are nutritionally well balanced, and contain high levels of moisture, proteins, fat, energy, ash, carbohydrates, reducing sugars, essential n‐3 fatty acids and good ratios of polyunsaturated fatty acids (PUFA)/saturated fatty acids. In fact, the PUFA represent 66% of the total fatty acids. The samples had good microbiological quality, as none of the samples had toxigenic species.
α‐Lactalbumin solubilisation from a precipitated whey protein concentrates fraction: pH and calcium concentration effects
Authors: Fernández Ayoa , Menéndez Violeta , Riera Francisco A.
Published: December 2011

The selective precipitation of α‐lactalbumin (α‐La) is the basis for one of the possible methods in whey protein fractionation. Calcium concentration, type of acid added and pH play important roles in α‐La precipitation and on the following resolubilisation. Two washing steps are enough for quantitative removal of β‐lactoglobulin entrapped in the precipitate. α‐La losses are minimised during washing steps (5%) when NaCl is used as washing agent. The most important parameter to control during the resolubilisation step is pH, the maximum amount of the initial re‐dissolved α‐La being 76% when the pH is adjusted to 7.5, CaCl2 concentration is 0.2 m and prior precipitation is carried out adding citric acid. Addition of CaCl2 is not necessary to dissolve α‐La because of the fact that there is enough calcium in the precipitate to join all α‐La; however, its presence improves the solubilisation yield (66% vs. 75%).
Surgical nurses’ perceptions of ethical dilemmas, moral distress and quality of care
Authors: DeKeyser Ganz Freda , Berkovitz Keren
Published: December 2011

dekeyser ganz f. & berkovitz k. (2012) Surgical nurses’ perceptions of ethical dilemmas, moral distress and quality of care. Journal of Advanced Nursing68(7), 1516–1525.

Aim.  To describe surgical nurses’ perceived levels of ethical dilemmas, moral distress and perceived quality of care and the associations among them.

Background.  Nurses are committed to providing quality care. They can experience ethical dilemmas and moral distress while providing patient care. Little research has focused on the effect of moral distress or ethical dilemmas on perceived quality of care.

Design.  Descriptive, cross‐sectional study.

Method.  After administration and institutional Research Ethics Committee approval, a researcher requested 119 surgical nurses working in two Israeli hospitals to fill out three questionnaires (personal background characteristics; Ethical Dilemmas in Nursing and Quality of Nursing Care). Data collection took place from August 2007 to January 2008.

Results.  Participant mean age was 39·7 years. The sample consisted mostly of women, Jewish and married staff nurses. The majority of nurses reported low to moderate levels of ethical dilemma frequency but intermediate levels of ethical dilemma intensity. Frequency of ethical dilemmas was negatively correlated with level of nursing skill, meeting patient’s needs and total quality of care. No important correlations were found between intensity of ethical dilemmas and quality of care.

Conclusions.  Levels of ethical dilemma frequency were higher than intensity. Nurses tended to be satisfied with their level of quality of care. Increased frequency of ethical dilemmas was associated with some aspects of perceived quality of care.

Relevance to clinical practice.  Quality of care is related to ethical dilemmas and moral distress among surgical nurses. Therefore, efforts should be made to decrease the frequency of these feelings to improve the quality of patient care.
Social pleiotropy and the molecular evolution of honey bee vitellogenin
Authors: HAVUKAINEN HELI , HALSKAU ØYVIND , AMDAM GRO V.
Published: December 2011

In this issue of Molecular Ecology, Kent et al. (2011) describe the adaptive evolution of honey bee vitellogenin that belongs to a phylogenetically conserved group of egg yolk precursors. This glyco‐lipoprotein leads a double life: it is central to egg production in the reproductive queen caste, and a regulator of social behaviour in the sterile worker caste. Does such social pleiotropy constrain molecular evolution? To the contrary; Kent et al. show that the vitellogenin gene is under strong positive selection in honey bees. Rapid change has taken place in specific protein regions, shedding light on the evolution of novel vitellogenin functions.
Water avoidance stress induces frequency through cyclooxygenase‐2 expression: A bladder rat model
Authors: Yamamoto Keisuke , Takao Tetsuya , Nakayama Jiro , Kiuchi Hiroshi , Okuda Hidenobu , Fukuhara Shinichiro , Yoshioka Iwao , Matsuoka Yasuhiro , Miyagawa Yasushi , Tsujimura Akira , Nonomura Norio
Published: December 2011

Objectives:  Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase‐2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase‐2 expression in a rat bladder model of water avoidance stress.

Methods:  After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase‐2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase‐2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase‐2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting.

Results:  Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase‐2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase‐2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase‐2 expression exclusively in smooth muscle cells. All water avoidance stress‐induced changes were reduced by cyclooxygenase‐2 inhibitor pretreatment.

Conclusions:  Chronic stress might cause frequency through cyclooxygenase‐2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase‐2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis.
Intra‐ and postoperative adverse events in children with nephrotic syndrome requiring surgery under general anesthesia
Authors: Greff Bruno , Faivre Judith , Carli Pierre A. , Niaudet Patrick , Orliaguet Gilles A.
Published: December 2011

Background:  There are few data regarding perioperative adverse events in children with nephrotic syndrome.

Objectives:  The aim of this study was to describe the nature and frequency of perioperative adverse events in children with nephrotic syndrome.

Materials and Methods:  This is a retrospective study from a large university pediatric hospital. All procedures under general anesthesia in children with nephrotic syndrome between January 1995 and May 2007 were included, with the exception of renal transplantation. Data were collected on demographics, etiology of nephrotic syndrome and related treatments, surgical procedures and anesthetic techniques, and pre‐ and postoperative treatments. Adverse events occurring during the intraoperative period and up to the fifth postoperative day were recorded.

Results:  Data on eight patients who underwent 24 surgical or interventional procedures under general anesthesia over the study period were reviewed. Three patients had steroid‐resistant nephrotic syndrome and five patients had congenital or infantile nephrotic syndrome. Five patients had progressed to end‐stage renal failure requiring dialysis. General anesthesia was performed for: nephrectomy (n = 9), central venous catheter insertion (n = 8), peritoneal dialysis catheter insertion (n = 5), and emergency surgery in two cases (acute intestinal intussusception and hemodialysis catheter insertion). Three patients were receiving aspirin and one anticoagulant therapy. No postoperative thrombosis or infections, bleeding, peripheral edema or ascites, and increase in kalemia were noted. There was no significant postoperative increase in median serum creatinine level.

Conclusions:  Surgical procedures were seldom associated with the occurrence of perioperative adverse events. However, larger studies are needed to confirm these results.