bluuebunny/crossref_metadata_2025 huggingface dataset:
35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE
Authors: Landschoot G. V. , Savy V. , Canel N. , Ferraris S. , Salamone D.
Published: December 2016

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P < 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT.

Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells
40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION
Authors: Appeltant R. , Somfai T. , Santos E. C. S. , Kikuchi K.
Published: December 2016

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL−1 cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL−1 polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL−1 eCG, 10 IU mL−1 hCG, 1 mM dibutyryl cAMP, and 10 ng mL−1 epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification.
R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).
54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY
Authors: Nongbua T. , Utta A. , Am-In N. , Suwimonteerabutr J. , Johannisson A. , Morrell J.
Published: December 2016

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.
72 EFFECT OF PROGESTERONE SUPPLEMENTATION ON DAY 4 AFTER TIMED ARTIFICIAL INSEMINATION ON PREGNANCY RATE OF LACTATING DAIRY COWS
Authors: Tortorelli G. , Azrak A. J. , da Costa Andrade V. , dos Santos Ramos R. , Moraes A. S. , da Silva Menon M. A. , Bastos M. R. , da Costa Carrer C.
Published: December 2016

Progesterone plays an important role in the maintenance of pregnancy. It is hypothesised that insufficient progesterone early in pregnancy may result in embryonic loss, and that supplemental progesterone would decrease pregnancy loss in dairy cows. The aim of this study was to compare the effect of progesterone supplementation after timed AI on pregnancy rate of dairy cows. The experiment was conducted in a single commercial herd, in Sao Paulo State (Brazil), during a period of 1 year (January–December 2016). The cows were assigned at a nonpregnancy diagnosis 32 ± 3 days after AI to 1 to 6 resynchronization treatments. They were submitted to the same fixed time AI protocol: Day 0, 0.1 mg of gonadorelin, 2 mg of oestradiol benzoate, and an intravaginal device of 1.9 g of progesterone; Day 7, 25 mg of dinoprost tromethamine; Day 8, 25 mg of dinoprost tromethamine, 1 mg of oestradiol cipionate, and device withdrawal; Day 10, AI. At Day 14, the cows were alternately arranged in 2 groups. Group 1 received a 900-mg progesterone supplementation IM injection, and Group 2 did not receive progesterone supplementation. Then, 32 ± 3 days after insemination, pregnancy diagnosis was performed by transrectal ultrasonography, and the data were tabulated. Full statistical analysis will be done at the end of the study, when more than 500 cows will be allocated to the 2 groups. From January until April 2016, 230 lactating dairy cows were submitted to the experiment. Both groups had similar average days in lactation (166), average number of lactations (1.95), and average number of inseminations (4.3). Group 1 had a pregnancy rate of 25% (29/116), and group 2 had a pregnancy rate of 15.8% (18/114) (P = 0.01, one-way ANOVA). Based on these preliminary data, pregnancy rates of lactating dairy cows may be improved with progesterone supplementation on Day 4 after insemination. These results need to be confirmed on larger numbers of cows.
85 REGULATION OF STEAROYL-CoENZYME A DESATURASE BY FATTY ACIDS IS ESSENTIAL TO PORCINE EARLY EMBRYO DEVELOPMENT
Authors: Lee D.-K. , Hwang J. Y. , Choi K.-H. , Kim S.-H. , Oh J.-N. , Lee C.-K.
Published: December 2016

Various fatty acids are found in large amounts in follicular fluid and the uterus. These fatty acids are known to regulate lipid metabolism in a mammal’s embryo. Lipid metabolism provides a source of energy, and its importance during embryogenesis is being increasingly recognised. Especially, the pig has larger amounts of intercellular lipid bilayers in the embryo than do other species, which indicates the porcine embryo is more dependent on fatty acid on their metabolic pathway. This study investigated the transcriptome analysis data of in vivo embryos and the effect of oleic acid (C18:1n-9) and stearic acid (C18:0) on in vitro-produced porcine embryos. In transcriptome analysis of in vivo embryos, we found several genes were increasing before and after maternal zygotic transition stage, and interactions were mapped and given a significance score. Among these genes, stearoyl-coenzyme A desaturase (SCD) gene was significantly increased during 8-cell to blastocyst stage. Stearoyl-coenzyme A desaturase is responsible for converting saturated fatty acid (stearic acid) to monounsaturated fatty acid (oleic acid). Furthermore, we treated with oleic acid (50, 100, 250, and 500 μM) and stearic acid (50, 75, and 100 μM) on 2 day and 4 day after parthenogenetic embryos. Both oleic acid and stearic acid concentration over 100 μM had a negative effect on blastocyst formation rate and cell numbers because of exogenous fatty acid toxicity. In addition, there was no significant difference among all stearic acid treated groups and total cell number of oleic acid treated blastocyst. However, the 2-day oleic acid treated group (45.92 ± 4.01, n = 8, 2 day, 100 μM) had a significant increase of blastocyst formation rate (P < 0.05) compared with the control group (34.88 ± 2.93, n = 8). These data could support that porcine embryos can use exogenous oleic acid as a metabolic energy source. The data also demonstrate the important role of SCD in porcine early embryo development. To confirm the transcriptome analysis, we are investigating mRNA level of SCD on in vitro-produced embryos at 1-cell to morula and blastocyst stage on the control group, oleic acid treated group, fetal bovine serum treated group, and nontreated group. Furthermore, we are investigating SCD inhibition assay by A939572 (SCD inhibitor) on parthenogenetic embryos in a fetal bovine serum treated group and nontreated group for identifying blastocyst formation rate and total cell numbers.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2055199) and Next BioGreen 21 program (PJ0113002016), Rural Development Administration, Republic of Korea.
90 OVIDUCTAL EPITHELIAL CELLS Co-CULTURE PROMOTES GOAT (CAPRA HIRCUS) IN VITRO PARTHENOGENETIC EMBRYO DEVELOPMENT
Authors: Mahajan M. , Nagoorvali D. , Rawat N. , Chauhan M. S. , Manik R. S. , Singla S. K. , Palta P. , Singh M. K.
Published: December 2016

Co-culture of pre-implantation embryos with oviducal epithelial cells mimics the in vivo conditions, thus, playing a crucial role in embryo metabolism and gene expression and finally supporting embryonic developmental competence in several ways. Hence, the objective of the present study was to evaluate the effect of goat oviducal epithelial cells (GOEC) co-culture on goat parthenogenetic embryonic development, quality, and relative mRNA abundance of genes related to developmental competence and oxidative stress. The GOEC were obtained from goat oviducts by squeezing and thorough washing with TCM-199 + 10% fetal bovine serum. Goat cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in TCM-199 + 10% fetal bovine serum supplemented with 5 μg mL−1 of FSH, 10 μg mL−1 of LH, and 1 μg mL−1 of β-oestradiol for 27 h in CO2 incubator with 5% CO2 and at 38.5°C with >95% RH. In vitro matured cumulus–oocyte complexes were denuded and activated with 5 μM calcium ionophore and 2 mM 6-DMAP. Following activation, embryos were co-cultured with and without GOEC (control) in mCR2aa media. The blastocyst development rate was significantly (P < 0.05) higher (23.00 ± 1.15% v. 17.33 ± 1.45%) in the media cultured with GOEC than in control. The total cell number of blastocysts (n = 4) was also found to be significantly more (167.25 ± 17.51 v. 110.25 ± 12.02) than that of control (P < 0.05). However, the apoptotic index (3.76 ± 0.23% v. 7.97 ± 1.99%) was not significantly different in both groups. Further, RNA was isolated from both groups (20 each) of blastocysts on Day 8, and cDNA was prepared. Analysis by qPCR revealed that the relative mRNA abundance of development related genes, i.e. VEGF, BMP4, and CCNB1, showed significantly high (P < 0.05) expression, whereas the expression of CRABP1 was significantly low (P < 0.05) in GOEC co-culture than control. Oxidative stress related genes GPX-1 and SOD2 had comparable expression in both the culture systems, whereas a nonsignificant (P < 0.05) increase in expression of PRDX1 was observed in GOEC co-culture group. In conclusion, co-culture of embryos with GOEC in the simple culture media like mCR2aa helps in improving developmental competence and quality of parthenogenetic embryos.
This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.
Cognitive individual differences in second language learning and processing
Authors: Granena Gisela , Jackson Daniel O. , Yilmaz Yucel
Published: December 2016

In this introductory chapter, we first describe the rationale for this volume, linking cognitive individual differences (IDs) to previous research in second language acquisition (SLA), and other, related fields. Next, we present a definition of cognitive abilities and illustrate how differences in these abilities may have important implications for several strands of research, including those dealing with second language aptitude, instructed SLA, comprehension and production, novel SLA, bilingualism, cognitive training, and measurement. This collective volume contains 14 additional chapters offering multiple, interdisciplinary perspectives on how individuals process and acquire languages, which incorporate theoretical, methodological, and empirical advances in the study of cognitive IDs. In the last section of this chapter, we summarize these contributions and draw explicit connections between them. The chapter concludes with some thoughts on the practical significance of this volume, in terms of researching cognitive IDs and understanding their implications.
Foreign language aptitude, acquisitional sequences, and psycholinguistic processes
Authors: Skehan Peter
Published: December 2016

This chapter has two broad aims: to relate aptitude tests and recent aptitude research, including that reported in this volume, to proposed second language acquisition stages; and to relate aptitude testing to the contrasts between domain-generality and domain-specificity, on the one hand, and explicit and implicit processes, on the other. Regarding SLA stages, a broad distinction is made between stages concerned with developing knowledge and those concerned with developing control. It is argued that aptitude tests have concentrated on the first and that there is scope to develop more aptitude tests to assess the second. Regarding the two major contrasts, it is argued that analysing aptitude tests in these terms is useful as a means of indicating what underlying theories they have. In that respect it is interesting that the most recent major battery (HiLAB: domain-general, often implicit) contrasts with older batteries (e.g. MLAT: domain specific, explicit). It is argued that aptitude tests, viewed in this way, can make theoretical contributions to second language acquisition more generally in characterising what language learning abilities consist of.
Miniature natural language learning in L2 acquisition research
Authors: Kempe Vera , Brooks Patricia J.
Published: December 2016

Researchers who wish to study mechanisms underlying second language (L2) learning and factors influencing its success face a trade-off between low experimental control in studies of naturalistic language acquisition and low typological and ecological validity in studies of artificial language learning. The methodological solution we propose is a miniature natural language learning paradigm, which exposes participants to selected elements of an unfamiliar L2, in the form of meaningful dialogues, over a period of several laboratory sessions. We show how this methodology allows researchers to understand how various components of learning interact as learners are exposed to vocabulary and grammar simultaneously, and how characteristics of both the learner and the input influence learning outcomes.
Interrelationships among L2 linguistic knowledge, working memory functions, and L2 reading
Authors: Çeçen Sevdeğer , Erçetin Gülcan
Published: December 2016

The main purpose of the current study is to investigate whether L2 reading comprehension is related to the processing and storage functions of working memory (WM) as well as explicit and implicit sources of knowledge in the L2. Byproducts of this question involve (a) whether the relationship between WM and reading comprehension is attributable to the linguistic nature of the complex span task; (b) whether WM is related to explicit and implicit types of linguistic knowledge in the L2. Participants were 84 late adult learners of English with a B2 level of proficiency. WM capacity was measured through reading span tasks (RST) administered in L1 and L2 and an operation span task (OST). An untimed grammaticality judgment test (GJT) and a metalinguistic knowledge test (MKT) were administered to measure explicit linguistic knowledge in the L2 while a timed GJT and an elicited oral imitation test (EOI) were used to measure implicit linguistic knowledge. Reading comprehension scores were obtained from a retired paper-based TOEFL. A principal component analysis (PCA) on the independent variables revealed a three-component model where processing and storage scores obtained from span tasks loaded on separate components reflecting a distinction between processing and storage functions of WM whereas explicit and implicit L2 knowledge measures loaded together on a single component. A regression analysis performed with factor scores from the PCA as independent variables and L2 reading comprehension scores as the dependent variable revealed significant contributions of WM’s processing function and L2 linguistic knowledge to reading comprehension in the L2. This suggests that the variance shared by WM capacity and L2 reading comprehension is not due to the linguistic nature of the span task but to the general processing efficiency of verbal WM capacity.
Influence of martensitic transformation on welding residual stress in plates and pipes
Authors: Wang Xue , Hu Lei , Xu Qiang , Chen Dong-xu , Sun Song-tao
Published: December 2016

Martensitic filler metals with low transformation temperatures can efficiently mitigate harmful tensile welding residual stress. It is vital to clarify the difference in the influence of martensitic transformation for different shapes of welded joints. This article outlines the influence of martensitic transformation in a butt-welded plate and a butt-welded pipe which were designed to have the same dimensions of the cross-section perpendicular to weld line. A clear difference in the influence of martensitic transformation was found in these two joints. Longitudinal tensile stress in weld zone is efficiently reduced in both joints, whereas longitudinal tensile stress is formed in base metal near the weld zone in the pipe. A notably greater influence on transverse stress is found in the pipe than in the plate.
NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling
Authors: Masoumi Katarzyna Chmielarska , Daams Renée , Sime Wondossen , Siino Valentina , Ke Hengning , Levander Fredrik , Massoumi Ramin
Published: December 2016

The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells.
Diverse activities of viralcis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging
Authors: Pocock Ginger M. , Zimdars Laraine L. , Yuan Ming , Eliceiri Kevin W. , Ahlquist Paul , Sherer Nathan M.
Published: December 2016

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include “burst” RNA nuclear export dynamics regulated by HIV-1’s Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element–specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation.
Addressing Beacon re-identification attacks: quantification and mitigation of privacy risks
Authors: Raisaro Jean Louis , Tramèr Florian , Ji Zhanglong , Bu Diyue , Zhao Yongan , Carey Knox , Lloyd David , Sofia Heidi , Baker Dixie , Flicek Paul , Shringarpure Suyash , Bustamante Carlos , Wang Shuang , Jiang Xiaoqian , Ohno-Machado Lucila , Tang Haixu , Wang XiaoFeng , Hubaux Jean-Pierre
Published: December 2016

The Global Alliance for Genomics and Health (GA4GH) created the Beacon Project as a means of testing the willingness of data holders to share genetic data in the simplest technical context—a query for the presence of a specified nucleotide at a given position within a chromosome. Each participating site (or “beacon”) is responsible for assuring that genomic data are exposed through the Beacon service only with the permission of the individual to whom the data pertains and in accordance with the GA4GH policy and standards.

While recognizing the inference risks associated with large-scale data aggregation, and the fact that some beacons contain sensitive phenotypic associations that increase privacy risk, the GA4GH adjudged the risk of re-identification based on the binary yes/no allele-presence query responses as acceptable. However, recent work demonstrated that, given a beacon with specific characteristics (including relatively small sample size and an adversary who possesses an individual’s whole genome sequence), the individual’s membership in a beacon can be inferred through repeated queries for variants present in the individual’s genome.

In this paper, we propose three practical strategies for reducing re-identification risks in beacons. The first two strategies manipulate the beacon such that the presence of rare alleles is obscured; the third strategy budgets the number of accesses per user for each individual genome. Using a beacon containing data from the 1000 Genomes Project, we demonstrate that the proposed strategies can effectively reduce re-identification risk in beacon-like datasets.
Lessons from characterization and treatment of the autoinflammatory syndromes
Authors: Aksentijevich Ivona , McDermott Michael F.
Published: December 2016

Purpose of review

The list of genes associated with systemic inflammatory diseases has been steadily growing because of the explosion of new genomic technologies. Significant advances in the past year have deepened our understanding of the molecular mechanisms linked to inflammation and elucidated insights on the efficacy of specific therapies for these and related conditions. We review the molecular pathogenesis of four recently characterized monogenic autoinflammatory diseases: haploinsufficiency of A20, otulipenia, a severe form of pyrin-associated disease, and a monogenic form of systemic juvenile idiopathic arthritis.

Recent findings

The scope of autoinflammation has been broadened to include defects in deubiquitination and cellular redox homeostasis. At the clinical level, we discuss the biological rationale for treatment with cytokine inhibitors and colchicine in respective conditions and the use of interleukin-1 antagonism for diagnostic and therapeutic purposes in the management of undifferentiated autoinflammatory disorders.

Gene discoveries coupled with studies of molecular function provide knowledge into the biology of inflammatory responses and form the basis for genomically informed therapies. Diseases of dysregulated ubiquitination constitute a novel category of human inflammatory disorders.
Predictors of Positive Margins at Time of Loop Electrosurgical Excision Procedure
Authors: Liss Jill , Alston Meredith , Krull Mona B. , Mazzoni Sara E.
Published: December 2016

Objective

The aim of the study was to identify factors associated with the presence of high-grade squamous intraepithelial lesion (HSIL) at surgical margins of loop electrosurgical excision procedure (LEEP) pathology specimens.

Materials and Methods

All women evaluated for lower genital tract disease at a single academic institution were prospectively entered into a database. The database was queried for all women who had a LEEP performed for indications within contemporary American Society of Colposcopy and Cervical Pathology guidelines between April 1, 2013, and April 30, 2015. Factors extracted from the database included demographics, contraception, weight, tobacco use, provider volume, resident participation, history of cervical procedure, and pathology features including preceding Pap test category. A positive margin was defined as HSIL on either the endocervical or ectocervical margin of the LEEP specimen or in endocervical curettage specimen after LEEP. We performed univariable statistics to identify factors associated with positive margins and then logistic regression modeling on significant factors for the outcome of positive margins.

Results

Two hundred sixty-nine women were identified. Seventy five (27.8%) of these women had positive margins. Only tobacco use, gravity, parity, and preceding Pap category were significant on univariable analysis. After multivariable analysis, smokers remained more likely to have positive margins (odds ratio = 2.01; CI = 1.12–3.6;p< .01) as did those with preceding HSIL Pap tests (odds ratio = 1.96; CI = 1.13–3.41;p< .01).

Conclusions

In our population, of all the factors assessed, only tobacco use and preceding high-grade Pap tests were associated with positive margins at time of LEEP. This information may be helpful in preprocedural planning to optimize treatment.
The Accuracy of Large Loop Excision of the Transformation Zone Specimen Dimensions in Determining Volume: A Multicentric Prospective Observational Study
Authors: Carcopino Xavier , Mancini Julien , Prendiville Walter , Gondry Jean , Chevreau Julien , Lamblin Gery , Atallah Anthony , Lavoue Vincent , Caradec Claire , Baldauf Jean-Jacques , Bryand Angélique , Henno Sébastien , Agostini Aubert , Douvier Serge , Jarniat Adelaide , Riethmuller Didier , Mendel Anne , Brun Jean-Luc , Rakotomahenina Hajanarina , Preaubert Lise
Published: December 2016

Objective

The aim of the study was to determine which mathematical formula of specimen dimensions is the most accurate method of determining volume of the excised specimen at loop excision of the transformation zone.

Materials and Methods

A multicenter prospective observational study was conducted. A total of 258 patients who had a loop excision of the transformation zone performed as treatment of cervical intraepithelial neoplasia 2–3 were included. The dimensions and the volume of the specimen were measured at the time of the procedure, before formaldehyde fixation. The volume was measured by immersing the specimen in a graduated cylinder using Archimedes fluid displacement technique. The measured volume was compared with the calculated volume using different volume formulas, that is, a cone, a cylinder, a parallelepiped, and a hemiellipsoid. The main outcome measure was the relationship between calculated volume (using the dimensions of thickness, length, and circumference) and the measured volume of the specimen.

Results

The mean (SD) thickness, length, and circumference of specimens were 8.8 mm (3.8), 12.7 mm (5.9), and 45.7 mm (16.8), respectively. The mean (SD) measured volume was 2.53 (1.49) mL. Using the formula for the volume of a cone, a cylinder, a parallelepiped and a hemiellipsoid, estimated volumes were 1.03 mL (1.22), 3.10 mL (3.65), 6.20 mL (7.31), and 2.07 mL (2.44), respectively. The highest intraclass correlation coefficient between measured and calculated volume was observed when using the formula for the volume of a hemiellipsoid specimen (0.47, 95% CI = 0.36–0.56).

Conclusions

The hemiellipsoid formula is the most accurate determinant of the excised volume. Other formulas do not allow for an accurate estimation of the excised volume.
Public Health Benefit of Peer-Referral Strategies for Detecting Undiagnosed HIV Infection Among High-Risk Heterosexuals in New York City
Authors: Gwadz Marya , Cleland Charles M. , Perlman David C. , Hagan Holly , Jenness Samuel M. , Leonard Noelle R. , Ritchie Amanda S. , Kutnick Alexandra
Published: December 2016

Identifying undiagnosed HIV infection is necessary for the elimination of HIV transmission in the United States. The present study evaluated the efficacy of 3 community-based approaches for uncovering undiagnosed HIV among heterosexuals at high-risk (HHR), who are mainly African American/Black and Hispanic. Heterosexuals comprise 24% of newly reported HIV infections in the United States, but experience complex multilevel barriers to HIV testing. We recruited African American/Black and Hispanic HHR in a discrete urban area with both elevated HIV prevalence and poverty rates. Approaches tested were (1) respondent-driven sampling (RDS) and confidential HIV testing in 2 sessions (n = 3116); (2) RDS and anonymous HIV testing in one session (n = 498); and (3) venue-based sampling (VBS) and HIV testing in a single session (n = 403). The main outcome was newly diagnosed HIV infection. RDS with anonymous testing and one session reached HHR with less HIV testing experience and more risk factors than the other approaches. Furthermore, RDS with anonymous (4.0%) and confidential (1.0%) testing yielded significantly higher rates of newly diagnosed HIV than VBS (0.3%). Thus peer-referral approaches were more efficacious than VBS for uncovering HHR with undiagnosed HIV, particularly a single-session/anonymous strategy, and have a vital role to play in efforts to eliminate HIV transmission.
Frailty and Circulating Markers of Inflammation in HIV+ and HIV− Men in the Multicenter AIDS Cohort Study
Authors: Margolick Joseph B. , Bream Jay H. , Martínez-Maza Otoniel , Lopez Joe , Li Xiuhong , Phair John P. , Koletar Susan L. , Jacobson Lisa P.
Published: December 2016

Background:

Frailty is associated with immune activation and inflammation in the elderly general population, but whether this is true in the younger HIV-infected (HIV+) population is not known.

Methods:

We analyzed 24 serologic biomarkers of monocyte, T-cell, or B-cell activation in HIV− (n = 207) and HIV+ (n = 714; 75% virologically suppressed) men who have sex with men in the Multicenter AIDS Cohort Study (MACS) and were classified as frail or nonfrail according to expression or nonexpression of the frailty phenotype at 2 consecutive study visits.

Results:

After correction for multiple comparisons and adjustment for age, race, study site, and education, frailty in HIV+ men was significantly (P< 0.002) associated with higher levels of sCD14, sIL2Rα, sTNF-R2, IL-6, and TNF-α; the association with higher levels of C-reactive protein (CRP) approached significance (P= 0.003). After further adjustment for body mass index (BMI), smoking, and comorbidities, only the association with C-reactive protein was significant atP< 0.002, with levels approximately 50% higher in frail compared with nonfrail men. These conclusions were not altered by restricting the analysis to HIV+ men who were virologically suppressed. Among HIV− men, none of these markers differed significantly by frailty.

Conclusions:

These data suggest that frailty in virologically suppressed HIV+ men was associated with immune activation beyond that due to treated HIV infection. The inflammatory markers associated with frailty were primarily products of activated monocytes/macrophages. Much, but not all, activation was accounted for by harmful behaviors and comorbidities. However, C-reactive protein, which is regulated by IL-6, was elevated in HIV+ frail men independent of these factors.
Prescription of Postexposure Prophylaxis for HIV-1 in the Emergency Room: Correct Transmission Risk Assessment Remains Challenging
Authors: Marzel Alex , Heinrich Henriette , Schilliger Lukas , Fehr Jan S. , Günthard Huldrych F. , Kouyos Roger , Rampini Silvana K.
Published: December 2016

Background:

Limited data are available about the accuracy of postexposure prophylaxis (PEP) prescription in the emergency rooms. Here, we evaluated PEP prescription decision making with respect to the risk of sexual HIV transmission and the exposed person's fear vis-à-vis HIV.

Methods:

Using a risk assessment algorithm, we retrospectively evaluated the adequacy of PEP prescription for all persons presenting at the emergency room of the University Hospital Zurich after consensual sex from 2007 to 2013. We used logistic regression to identify factors that correlate with risk-concordant and risk-discordant decisions.

Results:

We documented 975 persons with a total of 1051 visits for PEP: 83% were men, 71% were Swiss, and 37% were men who have sex with men. In 74% of visits, the decisions were concordant with the risk evaluation algorithm (22% discordant, 4% unknown). In 61% (644/1051) PEP was prescribed; however, in 12% (76/644) the prescriptions were without indication of HIV transmission risk and were attributed to the exposed person's request. Importantly, in 10% (101/1051) of all visits, there were potential risks but PEP was not prescribed, either because of physician's decision or exposed person's refusal. The presence of the source partner strongly correlated with appropriately withholding PEP (adjusted odds ratio for giving PEP 0.05; 95% confidence interval: 0.03 to 0.08).

Conclusions:

We found that 22% of PEP decisions were risk discordant because of exposed person's request, incorrect estimation of the sexual transmission risk by the physician, or exposed person's refusal to accept PEP. Emergency physicians may benefit from specialized risk assessment training and patients from education in HIV transmission risk awareness.
Optimal blood pressure target in stroke prevention
Authors: Yannoutsos Alexandra , Dreyfuss Tubiana Céline , Safar Michel E. , Blacher Jacques
Published: December 2016

Purpose of review

A generalized single blood pressure (BP) goal may appear not suitable to achieve an optimal stroke prevention at the individual level. The aim of the present review is to consider the clinical relevance of individualized BP goal according to patient's age, comorbidities and established cerebrovascular disease.

Recent findings

Observational and interventional data support heterogeneity in target organ prevention in that lower BP values than recommended by hypertension guidelines are associated with further stroke risk reduction. However, more ambitious BP treatment targets appear not broadly applicable to ageing hypertensive individuals or patients with established cerebrovascular disease.

Strict BP control in early diagnosed hypertensive individuals, without established arterial disease, should be a priority in primary stroke prevention. In ageing hypertensive individuals, difficult to control SBP can be considered as a marker of increased systemic arterial disease in the brain, the heart and the kidneys. A delayed aggressive intervention to reduce BP in these high-risk patients may be associated with a J-shaped relationship between BP level and adverse events. Moreover, patients suffering from stroke frequently presented with a high burden of small cerebral vessel lesions that are associated with an increased risk of cognitive decline progression, particularly with stringent BP levels.
Timing of head movements is consistent with energy minimization in walking ungulates
Authors: Loscher David M. , Meyer Fiete , Kracht Kerstin , Nyakatura John A.
Published: December 2016

Many ungulates show a conspicuous nodding motion of the head when walking. Until now, the functional significance of this behaviour remained unclear. Combiningin vivokinematics of quadrupedal mammals with a computer model, we show that the timing of vertical displacements of the head and neck is consistent with minimizing energy expenditure for carrying these body parts in an inverted pendulum walking gait. Varying the timing of head movements in the model resulted in increased metabolic cost estimate for carrying the head and neck of up to 63%. Oscillations of the head–neck unit result in weight force oscillations transmitted to the forelimbs. Advantageous timing increases the load in single support phases, in which redirecting the trajectory of the centre of mass (COM) is thought to be energetically inexpensive. During double support, in which—according to collision mechanics—directional changes of the impulse of the COM are expensive, the observed timing decreases the load. Because the head and neck comprise approximately 10% of body mass, the effect shown here should also affect the animals' overall energy expenditure. This mechanism, working analogously in high-tech backpacks for energy-saving load carriage, is widespread in ungulates, and provides insight into how animals economize locomotion.
Glyceraldehyde-3-Phosphate Dehydrogenase and Seven in Absentia Homolog -1 Interactions in Mammalian Cells Visualized Using Proximity Ligation Assay
Authors: Singh Harkewal , Melm Christopher
Published: December 2016

Proteins seldom function in isolation and thus protein-protein interactions are critical in understanding the molecular basis of diseases and health (1,2). There are several well established techniques that are used to investigate protein-protein interactions(3). Most of the methods require some form of genetic modification of the target protein and thus always adds extra steps. However, Proximity Ligation Assay(4-6) (PLA)akaDuolink® is one such method that requires no genetic modification of the target protein and probes protein-protein interactions in fixed live cells and tissues. Briefly, PLA requires the use of primary antibodies specific to the proteins of interest. Once the sample (fixed cells or tissues) is incubated with species specific primary antibodies, secondary antibodies that are conjugated with oligonucleotides (also known as PLUS and MINUS probes respectively) and connecter oligonucleotides are added. This complex is ligated if the two PLUS and MINUS probes are within 40nm of each other. The resulting nucleic acid is amplified using rolling circle amplification and then probed with appropriate fluorescent probes. If the two proteins are interacting, one could visualize the interaction as a single red foci (for example Far Red Detection) using fluorescent microscopy. Here, we used PLA to probe protein-protein interactions between Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and seven in absentia homolog -1 (Siah-1) – an E3 ubiquitin ligase. We first use PLA to show that GAPDH and Siah-1 proteins exist endogenously in the cytosol of multiple mammalian cell lines. Our data suggest the use of DU145 and T98G cell lines to show translocation of the GAPDH-Siah- 1 complex. Next, we used common nitrosylation agents(7,8) (S-nitrosoglutathione-GSNO and S-Nitroso-N-acetyl-DL-pencillamine–SNAP) in different concentrations and observed that GAPDH and Siah-1 interact presumably due to the nitrosylation of the former, which is consistent with previous studies(9,10). Interestingly, no interactions were observed between the two proteins in the absence of GSNO or SNAP indicating that nitrosylation might be critical for GAPDH-Siah1 interactions. Our results suggest that GAPDH-Siah-1 interactions originate in the cytosol and migrate to the nucleus under the conditions tested. We quantify the PLA signal using Duolink® Image Tool and observe a clear enhancement of GAPDH-Siah-1 PLA signal upon treating the cells with GSNO or SNAP. Next, we used R-(-)-Deprenyl (deprenyl), a known inhibitor of GAPDH4, and show that it abrogates GAPDH-Siah-1 PLA complex under the conditions tested. Finally, our data suggest that PLA can detect and quantify the GAPDH-Siah1 complex; a well-known protein-protein interaction implicated in neurodegeneration(9-11) and thus could be a method of choice for similar applications.
The effect of known mutator alleles in a cluster of clinicalSaccharomyces cerevisiaestrains
Authors: Skelly Daniel A. , Magwene Paul M. , Meeks Brianna , Murphy Helen A.
Published: December 2016

Natural selection has the potential to act on all phenotypes, including genomic mutation rate. Classic evolutionary theory predicts that in asexual populations, mutator alleles, which cause high mutation rates, can fix due to linkage with beneficial mutations. This phenomenon has been demonstrated experimentally and may explain the frequency of mutators found in bacterial pathogens. In contrast, in sexual populations, recombination decouples mutator alleles from beneficial mutations, preventing mutator fixation. In the facultatively sexual yeastSaccharomyces cerevisiae, segregating alleles ofMLH1andPMS1have been shown to be incompatible, causing a high mutation rate when combined. These alleles had never been found together naturally, but were recently discovered in a cluster of clinical isolates. Here we report that the incompatible mutator allele combination only marginally elevates mutation rate in these clinical strains. Genomic and phylogenetic analyses provide no evidence of a historically elevated mutation rate. We conclude that the effect of the mutator alleles is dampened by background genetic modifiers. Thus, the relationship between mutation rate and microbial pathogenicity may be more complex than once thought. Our findings provide rare observational evidence that supports evolutionary theory suggesting that sexual organisms are unlikely to harbor alleles that increase their genomic mutation rate.