bluuebunny/crossref_metadata_2025 huggingface dataset:
78 DIFFERENT EXTENDERS TO HARVEST EQUINE EPIDIDYMAL SPERM AND THEIR INFLUENCE ON FREEZABILITY
Authors: Soares R. F. , Papa F. O. , Magalhães L. C. O. , Monteiro G. A. , Martin I. , Dell'Aqua J. A. , Rocha A. S. , Melo-Oña C. M.
Published: December 2012

Harvesting and freezing epididymal sperm is a technology that enables the preservation of the gene pool from animals that had died either unexpectedly or because of colic conditions. This technique may also be employed in animals that have to be euthanized because of traumatic injuries. Therefore, the objective of the present study was to improve the process of freezing epididymal sperm using a freezing extender without the prior centrifugation of the samples. Twelve stallions aging between 3 and 6 years and of different breeds were used (Quarter Horse, Mangalarga, and Brazilian Jumping Horse). Stallions were castrated, and the cauda epididymides were isolated from the testis. The connective tissue was carefully dissected, and the cauda epididymides were straightened. A 200-µL pipette tip was attached to a 20-mL syringe, and the cauda epididymides were flushed using 40 mL of either (A) BotuSemen® (Nidacon, Mölndal, Sweden) or (B) BotuCrio® (Nidacon). They were then immediately processed at room temperature (25°C). Samples flushed with B were randomly subjected to either of the 2 procedures: B1) directly loaded into 0.5-mL straws or B2) centrifuged at 600g for 10 min, the supernatant was discarded, and the pellet was resuspended with B and loaded into 0.5-mL straws. The straws were kept at 5°C for 20 minutes followed by another 20 min at 6 cm above liquid nitrogen before immersion. After thawing at 46°C for 20 s the samples were analyzed by computer-assisted semen analysis (HTM – IVOS 12) and plasma membrane integrity was assessed using fluorescent probes (carboxyfluorescein diacetate and propidium iodide). Data were analyzed by ANOVA followed by the Tukey test (P < 0.05). No differences were observed for the values of total motility (A: 57.1 ± 12.35, B1: 46.3 ± 10.0, B2: 47.2 ± 12.84), progressive motility (A: 25.5 ± 9.05, B1: 21.7 ± 9.02, B2: 20.7 ± 7.20), percentage of rapids (A: 40.6 ± 15.92, B1: 30.7 ± 10.51, B2: 32.6 ± 12.39), and plasma membrane integrity (A: 47.8 ± 9.56, B1: 45.0 ± 13.81, B2: 41.3 ± 8.74). It was found that the fluid derived from epididymal secretions, which composes seminal plasma, had no influence on sperm parameters, because there was no difference among freezing protocols. Therefore, flushing equine epididymal cauda with B and freezing the samples without centrifuging can be successfully used. Both extenders (A and B) were efficient in protecting epididymal sperm throughout the freezing process.
83 IMPROVED SURVIVAL TO ONE-STEP REHYDRATION OF VITRIFIED - WARMED VERSUS FROZEN - THAWED IN VITRO-PRODUCED BOVINE BLASTOCYSTS
Authors: Trigal B. , Gómez E. , Caamaño J. N. , Muñoz M. , Correia E. , Martín D. , Carrocera S. , Díez C.
Published: December 2012

Vitrification allows cryopreservation of embryos while avoiding the detrimental effects derived from the intracellular ice formation during slow freezing. However, while slow freezing allows direct transfer of embryos, vitrification usually requires 1 or 2 rehydration steps after warming. The aim of this work was to analyze survival rates and quality of vitrified or slow frozen in vitro-produced (IVP) embryos, after warming/thawing by one-step procedures. Bovine blastocysts were produced in vitro, and on Day 7 and 8 excellent- and good-quality expanded blastocysts were selected for slow freezing (n = 175) or vitrification (n = 176) in 4 replicates. Slow freezing was performed in phosphate buffered saline containing ethylene glycol (1.5 M) and sucrose (0.1 M). Embryos were placed in a Biocool chamber (Biocool II, FTS® Systems Inc.) at –7°C for 5 min and seeded. After 5 min, embryos were cooled at –0.3°C min–1 until –32°C and plunged in LN2. Embryos were thawed in a water bath at 37°C for 30 s. Vitrification was performed in fibreplugs as previously described (Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). For warming, embryos were incubated for 5 min in TCM199–HEPES, 20% FCS, and 0.25 M sucrose. Thawed and warmed embryos were washed and subsequently cultured in mSOFaaci + 6 g L–1 BSA + 10% FCS for 48 h. Re-expansion (RE) (at 2, 24, and 48 h in culture) and hatching rates (HR; at 24 and 48 h in culture) were recorded. Total cells were counted in blastocysts that hatched at 24 and 48 h after fixation and bisbenzimide staining. Data were analyzed by ANOVA and are presented as least squares means ± standard error. No differences were found within RE at 2 and 24 h, and HR at 24 h (2-h RE: 94.1 ± 4.9 v. 95.4 ± 4.9; 24-h RE: 92.6 ± 4.9 v. 94.5 ± 4.9; HR: 21.0 ± 7.0 v. 20.6 ± 7.0, for slow frozen and vitrified embryos, respectively; P > 0.05). However, at 48 h, vitrified embryos hatched at higher rates than did slow-frozen embryos (53.6 ± 10.2 v. 32.5 ± 10.2; P < 0.05). Vitrified embryos (143.5 ± 12.7) had higher (P < 0.05) cell numbers than did slow-frozen embryos (106.1 ± 9.6) after hatching. Our results show that vitrification of IVP embryos in fibreplugs followed by a one-step warming is a promising candidate procedure to replace slow freezing for direct transfer on field. These results must be completed with embryo transfers to analyze pregnancy rates.
RTA2011-00090 (FEDER-INIA) is acknowledged. Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.
85 SYNERGISTIC EFFECT OF DEHYDRATION AND LOW TEMPERATURE ON THE SURVIVAL OF PANGASIUS CATFISH (PANGASIANODON HYPOPHTHALMUS) EMBRYOS
Authors: Bui X. N. , Nguyen T. H. , Nguyen T. N. , Nguyen V. H. , Nguyen T. T. , Nga Ha T. N. , Nguyen T. H. , Nguyen V. L. , Nguyen T. U.
Published: December 2012

Embryo cryopreservation is an important need for industrial catfish production, as well as for biodiversity conservation. However, due to the limit of membrane permeability and dehydration in fish embryos, no successful cryopreservation method has been reported. We present herein results of the effect of dehydration and low temperature on the survival of catfish embryos, to understand their cryobiological behaviour and develop an efficient freeze method for this species. Embryos at blastocyst stage were collected at 10 h after insemination and kept in a water tank with an air pump. Embryos with somites and optic cups clearly distinguished were selected and treated according to 4 treatment groups: group 1, control group: embryos were kept in water at room temperature for 2 h; group 2, treated for dehydration: embryos were kept in 1 M or 2 M sucrose solution for 2 h at room temperature; group 3, treated for low temperature: embryos were kept in water for 2 h at 0°C; and group 4, treated for dehydration and low temperature: embryos were kept in 1 M or 2 M sucrose solution for 2 h at 0°C. After treatments, all embryos were washed twice in water and incubated in separated water tanks for 2 days. The survival of embryo was evaluated by the rate of intact and hatched embryos during the first day after treatment. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. Our results showed that in the control group, all of the embryos were intact and hatched. In the group 2, the survival rate of embryos treated with 1 M sucrose was not different compared with the control group. However, in this group, no hatched embryos were obtained when treated with 2 M sucrose (Table 1). Also, no hatched embryos were observed when they were kept at 0°C without dehydration (group 3). A significant improving hatching rate was obtained in group 4 for embryos treated at 0°C and dehydrated in 2 M sucrose (90 v. 0% in group 3; P < 0.05). In conclusion, the combined treatment of dehydration (in 2 M sucrose concentration) and low temperature (at 0°C) can result in synergistic effect that are able to protect embryos from damages caused by treatment with either low temperature or dehydration conditions.

Table 1.Treated catfish embryos

This work was supported by a project from MARDVN.
88 VARIABLE DNA METHYLATION PROFILES AT IMPRINTED LOCI IN BOVINE EARLY PRE-IMPLANTATION EMBRYOS
Authors: O'Doherty A. M. , Magee D. , Beltman M. E. , Mamo S. , Rizos D. , Fair T.
Published: December 2012

The DNA methylation imprints, at maternally imprinted gene differentially methylated regions, are established during the postnatal growth stage of oogenesis, with paternal imprints being acquired in the perinatal prospermatagonia. Murine DNA methylation marks, at imprinted loci, are widely regarded to be resistant to post-fertilization demethylation events that occur in the paternal pronucleus of the zygote and to passive demethylation of the maternally derived genomic content from cleavage to the 16-cell stage. However, the DNA methylation profile of bovine imprinted genes following fertilization remains unknown. The objective of the current study was to analyze the methylation dynamics at several imprinted gene differentially methylated regions during bovine embryo development. In addition, a previously published RNA-seq database (Mamo et al. 2011 Biol. Reprod.) was mined for transcript abundance of genes associated with establishing and maintaining genomic imprints. Single in vivo blastocysts (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17), and implanting conceptii (Day 25) were collected (n = 4–9, per time point) from beef heifers. Genomic DNA was isolated and bisulfite modified, using the EZ DNA methylation direct kit (Zymo, Irvine, CA, USA), and used as template in bisulfite PCR reactions. The PCR products were verified by agarose gel electrophoresis and subsequently pyrosequenced. Observed methylation values were most highly variable in Day 7 blastocysts, with values ranging between 13 and 44% (IGF2R), 5 and 63% (PEG10), 7 and 59% (MEST), 3 and 61% (SNRPN), 12 and 64% (PLAGL1), and 20 and 32% (H19). There was a marked reduction in variability as embryonic development progressed, with values at Day 25 ranging from 37 to 41% (IGF2R), 34 to 38% (PEG10), 31 to 37% (MEST), 36 to 40% (SNRPN), 17 to 26% (PLAGL1), and 25 to 30% (H19). Statistical analysis (Levene’s test for equal variance) of methylation values for each gene at each time point confirmed that the methylation values observed in Day 7 embryos were significantly variable (P < 0.05) when compared with later developmental stages. Concordant with this finding, RNA transcript levels of associated methylation machinery genes DNMT3A, DNMT3B, and TRIM28 progressively increased from Day 7 to 13 and subsequently decreased from Day 13 to 16. Taken together our results demonstrate that in cattle DNA methylation marks, at imprinted loci, are highly variable at the blastocyst stage and are progressively stabilized with increasing days post-fertilization. This stabilization of imprint is coordinated with a window of increased levels of associated methylation machinery transcripts. Work presented here provides evidence of a novel mechanism for bovine embryonic DNA methylation imprint maintenance.
This work was funded by SFI grant number 07/SRC/B1156.
90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO
Authors: Caudle J. , Hamilton C. K. , Ashkar F. A. , King W. A.
Published: December 2012

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development.
Funding was provided by NSERC, the CRC program, and the OVC scholarship program.
91 SPERMATOZOA TELOMERE LENGTH DETERMINES EMBRYONIC TELOMERE LENGTH BEFORE EMBRYONIC GENOME ACTIVATION
Authors: de Frutos C. , Laguna-Barraza R. , Bermejo-Alvarez P. , Rizos D. , Gutierrez-Adan A.
Published: December 2012

A critical issue for species integrity is the existence of a telomere elongation program during embryogenesis that ensures sufficient telomere reserves in mammalian newborns. Two different mechanisms have been reported to act on telomere elongation during early embryogenesis: first, the telomerase, the ribonucleoprotein that adds telomeric repeats onto the chromosome ends, known to be responsible for the telomere lengthening at the morula-blastocyst transition in mice and bovine; second, in laboratory mice strains, mature oocytes increase the length of their relatively short telomeres between the 1-cell and 2-cell stages by a recombination or ALT-like pathway. In contrast, spermatozoa, the terminally differentiated male gametes, exhibit a very long telomere length (TL). The aim of this study was to clarify the potential role of the spermatozoa TL in the telomere lengthening occurring between oocyte and the 2-cell stage. For this purpose, we used 2 mouse species known to differ greatly in their TL [Mus musculus (hybrid C57CBAF1), long TL, and Mus spretus, short TL]. First, we compared relative TL in sperm samples from 5 age-matched males of each species by quantitative real-time PCR, with the numbers of telomere repeats being normalized, to the amount of DNA present in the sample (based on quantification of the Rn18S gene) by the comparative Ct method. Then, 1- and 2-cell embryos were produced by fertilizing Mus musculus oocytes with either Mus musculus or Mus spretus spermatozoa. The TL analysis in oocytes, zygotes, or 2-cell embryos was carried out by absolute quantification of telomere repeats by qPCR and normalized to the highest Ct observed value. Twenty to thirty samples per stage were analyzed, with each sample consisting in 2 matured oocytes, 2 zygotes, or one 2-cell embryo, to allow comparisons between stages. One-way ANOVA was used for statistical analysis. Mus spretus spermatozoa had significantly shorter telomeres than did Mus musculus (1.0 ± 0.1 v. 9.0 ± 1.5, respectively; P ≤ 0.01). The TL increased after fertilization from oocyte to zygote and 2-cell embryo stages in Mus musculus (1.0 ± 0.1, 1.5 ± 0.1, and 2.4 ± 0.2, respectively; P ≤ 0.01). In contrast, no differences were found in the TLs between the 3 stages in Mus spretus hybrids (oocyte: 1.0 ± 0.1; zygote: 1.0 ± 0.1; and 2-cell embryo: 1.0 ± 0.1), indicating that no elongation occurred after fertilization with spermatozoa with short telomeres. Herein, we demonstrated that before embryonic genome activation occurs, spermatozoa TL determines TL of the early embryo, suggesting that spermatozoon telomeres may act as recombination templates for early telomere lengthening right after syngamia.
95 ALTERED PROTEIN AND AMINO-ACID METABOLISM IN PREIMPLANTATION EMBRYOS FROM DIABETIC RABBITS
Authors: Gürke J. , Haucke E. , Thieme R. , Hirche F. , Schindler M. , Fischer B. , Navarrete Santos A.
Published: December 2012

During pregnancy the preimplantation period is a critical ontogenetic stage in embryo development. As the embryo is highly sensitive to its surrounding milieu and vulnerable to dysregulations by external stimuli, we investigated the influence of a maternal diabetes mellitus type 1 on blastocyst metabolism and protein modifications. Here we report on protein and amino acid metabolism in preimplantation embryos. A diabetes mellitus type 1 rabbit model was used to measure the protein and amino acid concentrations in blastocyst cavity fluid at Day 6 postcoitum. The protein concentration was enhanced in embryos from diabetic rabbits. The level of the various proteinogenic amino acids was unchanged except for the branched-chain amino acids (BCAAs; l-leucine, l-isoleucine, l-valine). The concentration of l-leucine [153.9 µM ± 60.14] and l-valine [231.95 µM ± 75.67] increased 2-fold and of l-isoleucine [99.85 µM ± 42.92] increased 3-fold. Due to the altered BCAA levels, we assumed a disturbed biodegradation. The expression of the BCAA oxidising enzymes (Bcat2, Bckdha, Dbt, and Dld) and BCAA transporters was determined by real-time PCR. Embryos grown in diabetic rabbits revealed a decreased expression of BCAA oxidizing enzymes and of the BCAA transporter LAT-2. The BCAA transporter LAT-2 was mainly localized in the embryoblast. The quality of proteins in blastocysts from diabetic rabbits was evaluated by analyzing protein glycation (advanced glycation end products, AGEs). Two specific AGE modifications, namely arg-pyrimidine and pentosidine, were detected in Day-6 blastocysts by Western Blot analysis. In blastocyst cavity fluid the AGE-specific fluorescence at 440ex/535em nm was significantly enhanced. Our findings show that not just the quantity of proteins but also the quality is affected in rabbit embryos grown in diabetic mothers. There is evidence to suggest that vital protein interactions and signalling pathways are misprogrammed with likely negative consequences for further life.
Supported by EU FP 7 EpiHealth (N°278418).
Neuronal integration in visual cortex elevates face category tuning to conscious face perception
Authors: Fahrenfort Johannes J. , Snijders Tineke M. , Heinen Klaartje , van Gaal Simon , Scholte H. Steven , Lamme Victor A. F.
Published: December 2012

The human brain has the extraordinary capability to transform cluttered sensory input into distinct object representations. For example, it is able to rapidly and seemingly without effort detect object categories in complex natural scenes. Surprisingly, category tuning is not sufficient to achieve conscious recognition of objects. What neural process beyond category extraction might elevate neural representations to the level where objects are consciously perceived? Here we show that visible and invisible faces produce similar category-selective responses in the ventral visual cortex. The pattern of neural activity evoked by visible faces could be used to decode the presence of invisible faces and vice versa. However, only visible faces caused extensive response enhancements and changes in neural oscillatory synchronization, as well as increased functional connectivity between higher and lower visual areas. We conclude that conscious face perception is more tightly linked to neural processes of sustained information integration and binding than to processes accommodating face category tuning.
Copper aerosols inhibit phytoplankton growth in the Mediterranean Sea
Authors: Jordi Antoni , Basterretxea Gotzon , Tovar-Sánchez Antonio , Alastuey Andrés , Querol Xavier
Published: December 2012

Aerosol deposition plays an important role in climate and biogeochemical cycles by supplying nutrients to the open ocean, in turn stimulating ocean productivity and carbon sequestration. Aerosol particles also contain elements such as copper (Cu) that are essential in trace amounts for phytoplankton physiology but that can be toxic at high concentrations. Although the toxicity of Cu associated with aerosols has been demonstrated in bioassay experiments, extrapolation of these laboratory results to natural conditions is not straightforward. This study provides observational evidence of the negative effect of aerosols containing high Cu concentrations on marine phytoplankton over a vast region of the western Mediterranean Sea. Direct aerosol measurements were combined with satellite observations, resulting in the detection of significant declines in phytoplankton biomass after atmospheric aerosol events characterized by high Cu concentrations. The declines were more evident during summer, when nanoflagellates predominate in the phytoplankton population and stratification and oligotrophic conditions prevail in the study region. Together with previous findings concerning atmospheric Cu deposition, these results demonstrate that the toxicity of Cu-rich aerosols can involve large areas of the world’s oceans. Moreover, they highlight the present vulnerability of oceanic ecosystems to Cu-rich aerosols of anthropogenic origins. Because anthropogenic emissions are increasing, large-scale negative effects on marine ecosystems can be anticipated.
DNA regions bound at low occupancy by transcription factors do not drive patterned reporter gene expression in
Drosophila
Authors: Fisher William W. , Li Jingyi Jessica , Hammonds Ann S. , Brown James B. , Pfeiffer Barret D. , Weiszmann Richard , MacArthur Stewart , Thomas Sean , Stamatoyannopoulos John A. , Eisen Michael B. , Bickel Peter J. , Biggin Mark D. , Celniker Susan E.
Published: December 2012

In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in
Drosophila
blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the
cis
-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional
cis
-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.
Genome-scale comparative analysis of gene fusions, gene fissions, and the fungal tree of life
Authors: Leonard Guy , Richards Thomas A.
Published: December 2012

During the course of evolution genes undergo both fusion and fission by which ORFs are joined or separated. These processes can amend gene function and represent an important factor in the evolution of protein interaction networks. Gene fusions have been suggested to be useful characters for identifying evolutionary relationships because they constitute synapomorphies or cladistic characters. To investigate the fidelity of gene-fusion characters, we developed an approach for identifying differentially distributed gene fusions among whole-genome datasets:fdfBLAST. Applying this tool to the Fungi, we identified 63 gene fusions present in two or more genomes. Using a combination of phylogenetic and comparative genomic analyses, we then investigated the evolution of these genes across 115 fungal genomes, testing each gene fusion for evidence of homoplasy, including gene fission, convergence, and horizontal gene transfer. These analyses demonstrated 110 gene-fission events. We then identified a minimum of three mechanisms that drive gene fission: separation, degeneration, and duplication. These data suggest that gene fission plays an important and hitherto underestimated role in gene evolution. Gene fusions therefore are highly labile characters, and their use for polarizing evolutionary relationships, without reference to gene and species phylogenies, is limited. Accounting for these considerable sources of homoplasy, we identified fusion characters that provide support for multiple nodes in the phylogeny of the Fungi, including relationships within the deeply derived flagellum-forming fungi (i.e., the chytrids).
A Pictet-Spengler ligation for protein chemical modification
Authors: Agarwal Paresh , van der Weijden Joep , Sletten Ellen M. , Rabuka David , Bertozzi Carolyn R.
Published: December 2012

Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C–C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.
Equatorial decline of reef corals during the last Pleistocene interglacial
Authors: Kiessling Wolfgang , Simpson Carl , Beck Brian , Mewis Heike , Pandolfi John M.
Published: December 2012

The Last Interglacial (LIG;
ca.
125,000 y ago) resulted from rapid global warming and reached global mean temperatures exceeding those of today. The LIG thus offers the opportunity to study how life may respond to future global warming. Using global occurrence databases and applying sampling-standardization, we compared reef coral diversity and distributions between the LIG and modern. Latitudinal diversity patterns are characterized by a tropical plateau today but were characterized by a pronounced equatorial trough during the LIG. This trough is governed by substantial range shifts away from the equator. Range shifts affected both leading and trailing edges of species range limits and were much more pronounced in the Northern Hemisphere than south of the equator. We argue that interglacial warming was responsible for the loss of equatorial diversity. Hemispheric differences in insolation during the LIG may explain the asymmetrical response. The equatorial retractions are surprisingly strong given that only small temperature changes have been reported in the LIG tropics. Our results suggest that the poleward range expansions of reef corals occurring with intensified global warming today may soon be followed by equatorial range retractions.
Development of the mammalian axial skeleton requires signaling through the Gα
i
subfamily of heterotrimeric G proteins
Authors: Plummer Nicholas W. , Spicher Karsten , Malphurs Jason , Akiyama Haruhiko , Abramowitz Joel , Nürnberg Bernd , Birnbaumer Lutz
Published: December 2012

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene
Gnai3
have fusions of ribs and lumbar vertebrae, indicating a requirement for Gα
i
(the “inhibitory” class of α-subunits) in somite derivatives. Mice with mutations of
Gnai1
or
Gnai2
have neither defect, but loss of both
Gnai3
and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in
Gnai3
/
Gnai1
double-mutant embryos, and crosses with a conditional allele of
Gnai2
indicate that Gα
i
is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.
Issues Regarding the Use of Sedatives in Fisheries and the Need for Immediate‐Release Options
Authors: Trushenski J. T. , Bowker J. D. , Cooke S. J. , Erdahl D. , Bell T. , MacMillan J. R. , Yanong R. P. , Hill J. E. , Fabrizio M. C. , Garvey J. E. , Sharon S.
Published: December 2012

The lack of an immediate‐release sedative (i.e., one for which no postsedation holding or withdrawal period is required) jeopardizes fish and fisheries research and poses considerable risk to those involved in aquatic resource management and the operation of public hatcheries and commercial fish farms. Carbon dioxide may be used as an immediate‐release sedative, but it is slow‐acting and difficult to apply uniformly and effectively. Tricaine methanesulfonate (MS‐222) is easier to apply but requires a 21‐d withdrawal period. The lack of an immediate‐release sedative approved by the U.S. Food and Drug Administration (FDA) is a consequence of numerous factors, including the complexities of the approval process, the substantial human and monetary resources involved, and the specialized nature of the work. Efforts are currently underway to demonstrate the safety and effectiveness of benzocaine‐ and eugenol‐based products as immediate‐release sedatives. However, pursuing approvals within the current framework will consume an exorbitant amount of public and private resources and will take years to complete, even though both compounds are “generally recognized as safe” for certain applications by the FDA. We recommend using risk management–based approaches to increase the efficiency of the drug approval process and the availability of safe and effective drugs, including immediate‐release sedatives, for use in the fisheries and aquaculture disciplines.
Patient- and Device-Specific Risk Factors for Peripherally Inserted Central Venous Catheter—Related Bloodstream Infections
Authors: Pongruangporn Makhawadee , Ajenjo M. Cristina , Russo Anthony J. , McMullen Kathleen M. , Robinson Catherine , Williams Robert C. , Warren David K.
Published: December 2012

Objective.

To determine the patient- and device-specific risk factors for hospital-acquired peripherally inserted central venous catheter–related bloodstream infections (PICC BSIs) in adult patients.

Design.

Nested case-control study.

Setting.

Barnes-Jewish Hospital, a 1,252-bed tertiary care teaching hospital.

Patients.

Adult patients with PICCs placed from January 1, 2006, through July 31, 2008.

Methods.

PICC BSI cases were identified using the National Healthcare Safety Network definition. Uninfected control patients with PICCs in place were randomly selected at a 3: 1 ratio. Patient- and device-related variables were examined using multivariate analysis.

Results.

The overall PICC BSI rate was 3.13 per 1,000 catheter-days. Independent risk factors for PICC BSIs included congestive heart failure (odds ratio [OR], 2.0 [95% confidence interval (CI), 1.26–3.17];P= .003), intra-abdominal perforation (OR, 5.66 [95% CI, 1.76–18.19];P= .004),Clostidium difficileinfection (OR, 2.25 [95% CI, 1.17–4.33];P= .02), recent chemotherapy (OR, 3.36 [95% CI, 1.15–9.78];P= .03), presence of tracheostomy (OR, 5.88 [95% CI, 2.99–11.55];P< .001), and type of catheter (OR for double lumen, 1.89 [95% CI, 1.15–3.10];P= .01; OR for triple lumen, 2.87 [95% CI, 1.39–5.92];P= .004). Underlying chronic obstructive pulmonary disease (OR, 0.48 [95% CI, 0.29–0.78];P= .03) and admission to surgical (OR, 0.43 [95% CI, 0.24–0.79];P= .006) or oncology and orthopedic (OR, 0.35 [95% CI, 0.13–0.99];P= .05) services were less likely to be associated with having a PICC BSI.

Conclusions.

We identified several novel factors related to PICC BSIs. These factors may inform preventive measures.
Characterization of disordered proteins with ENSEMBLE
Authors: Krzeminski Mickaël , Marsh Joseph A. , Neale Chris , Choy Wing-Yiu , Forman-Kay Julie D.
Published: December 2012

Summary: ENSEMBLE is a computational approach for determining a set of conformations that represents the structural ensemble of a disordered protein based on input experimental data. The disordered protein can be an unfolded or intrinsically disordered state. Here, we introduce the latest version of the program, which has been enhanced to facilitate its general release and includes an intuitive user interface, as well as new approaches to treat data and analyse results.

Availability and implementation: ENSEMBLE is a program implemented in C and embedded in a Perl wrapper. It is supported on main Linux distributions. Source codes and installation files, including a detailed example, can be freely downloaded at http://abragam.med.utoronto.ca/∼JFKlab.

Contact: forman@sickkids.ca

Supplementary information: Supplementary Material are available at Bioinformatics online.
Detection of the Rossiter–McLaughlin effect in the 2012 June 6 Venus transit
Authors: Molaro P. , Monaco L. , Barbieri M. , Zaggia S.
Published: December 2012

Eclipsing bodies on stars produce radial velocity variations on the photospheric stellar lines known as the Rossiter–McLaughlin (RM) effect. The body occults a small area of the stellar disc and, due to the rotation of the star, the stellar line profiles are distorted according to the projected location of the body on to the stellar disc. The effect originally observed in eclipsing binaries was also shown to be produced by extrasolar planets transits. Here we report the detection of the RM effect in the Sun due to the Venus transit of 2012 June 6. We used the integrated sunlight as reflected by the Moon at night time to record part of the transit by means of the high-precision HARPS spectrograph at the 3.6-m La Silla European Southern Observatory (ESO) telescope. The observations show that the partial Venus eclipse of the solar disc in correspondence of the passage in front of the receding hemisphere produced a modulation in the radial velocity with a negative amplitude of ≈−1 m s−1, in agreement with the theoretical model. The radial velocity change is comparable to the solar jitter and more than a factor of 2 smaller than previously detected in extrasolar hot Neptunes. This detection, facilitated by an amplification factor of 3.5 of the Venus radius due to proximity, anticipates the study of transits of Earth-size bodies in solar-type stars by means of a high-resolution spectrograph attached to a 40-m class telescope.
Evaluation of Eggplant Rootstocks and Pepper Varieties for Potential Resistance to Isolates of Phytophthora capsici from Michigan and New York
Authors: Foster J. M. , Naegele R. P. , Hausbeck M. K.
Published: December 2012

Phytophthora capsici is a soilborne pathogen of major economic importance in pepper, and of less importance in tomato and eggplant production. As soil fumigation becomes more expensive and limited, and fungicide insensitivity of P. capsici isolates becomes more prevalent, grafting is quickly becoming an industry-favored method to control soilborne diseases. Greenhouse experiments were performed to evaluate an eggplant cultivar (Classic), two eggplant lines (EG195, EG203), a pepper line (CM334), and three pepper cultivars (Paladin, Camelot, and Red Knight) for root rot resistance to 14 P. capsici isolates. The isolates showed various degrees of virulence between pepper and eggplant in both experiments. Both eggplant and one pepper lines showed moderate resistance to the most virulent isolates tested in experiment one. The partially resistant pepper cultivar, Paladin, was significantly more susceptible than CM334 and the eggplant lines, but was still resistant to most isolates. In the second experiment, the eggplant cultivar Classic and the susceptible pepper cultivar Red Knight were both susceptible to most isolates tested, while EG203 and EG195 were resistant to most isolates. The two eggplant breeding lines, EG195 and EG203, showed moderate resistance to all isolates tested in both experiments. This is the first reported evaluation of eggplant resistance to P. capsici. Further research is warranted to test eggplant lines EG195 and EG203 for resistance to a wide range of soilborne pests and to evaluate their usefulness as P. capsici–resistant rootstocks for peppers, tomatoes, and eggplants.
Spring Wheat Tolerance and Resistance to Heterodera avenae in the Pacific Northwest
Authors: Smiley Richard W. , Marshall Juliet M. , Gourlie Jennifer A. , Paulitz Timothy C. , Kandel Shyam L. , Pumphrey Michael O. , Garland-Campbell Kimberly , Yan Guiping , Anderson Monte D. , Flowers Michael D. , Jackson Chad A.
Published: December 2012

The cereal cyst nematode Heterodera avenae reduces wheat yields in the Pacific Northwest. Previous evaluations of cultivar resistance had been in controlled environments. Cultivar tolerance had not been evaluated. Seven spring wheat trials were conducted in naturally infested fields in three states over 2 years. A split-plot design was used for all trials. Five trials evaluated both tolerance and resistance in 1.8-by-9-m plots treated or not treated with nematicides. Two trials evaluated resistance in 1-m head rows where each wheat entry was paired with an adjacent row of a susceptible cultivar. Cultivars with the Cre1 resistance gene (‘Ouyen’ and ‘Chara’) reduced the postharvest density of H. avenae under field conditions, confirming Cre1 parents as useful for germplasm development. Ouyen was resistant but it was also intolerant, producing significantly lower grain yield in controls than in plots treated with nematicides. Susceptible cultivars varied in tolerance. Undefined resistance was identified in one commercial cultivar (‘WB-Rockland) and four breeding lines (UC1711, SO900163, SY-B041418, and SY-97621-05). This research was the first systematic field demonstration of potential benefits to be derived through development and deployment of cultivars with resistance plus tolerance to cereal cyst nematode in North America.
First Report of Downy Mildew Caused by Peronospora sordida on Butterflybush (Buddleja stachyoides) in Brazil
Authors: Albuquerque S. T. , Rocha F. B. , Barreto R. W.
Published: December 2012

Butterflybush (common name in Brazil, verbasco), Buddleja stachyoides Cham. & Schltdl. (Buddlejaceae), is an erect herb or small shrub, native to Brazil, that is listed both as a folk medicinal plant and as a pasture weed (4). In March 2012, a group of B. stachyoides plants growing in a pasture in Viçosa (state of Minas Gerais, Brazil), were found bearing typical downy mildew symptoms. The only pathogen reported associated with this plant species is Podosphaera xanthii (1) and because there is no record of downy mildew on members of Buddleja in Brazil, an investigation was carried out to clarify the pathogen identity. Diseased plants had lesions on living leaves that were vein-delimited, chlorotic, coalescing, and becoming necrotic adaxially and bearing downy mildew-like colonies over diseased tissues abaxially. The samples were dried in a plant press and a representative specimen was deposited in the local herbarium at the Universidade Federal de Viçosa (Accession No. VIC 31836). Reproductive structures were scraped from leaves with a scalpel and mounted in lactophenol. Preliminary observations indicated the pathogen as belonging to Peronospora (Peronosporaceae). The pathogen had the following morphology: Sporangiophores are 288 to 641 μm long, dichotomously branching up to seven times, hyaline, smooth, 5 to 16 μm wide at the trunk, branches 63 to 202 μm long; tips subacute, in pairs or rarely single, 5 to 19 μm long; sporangia subglobose to ellipsoidal, 12 to 22 × 11 to 17 μm, pale yellowish brown, non-papillate. Only one species of Peronospora is known to infect members of Buddleja, namely Peronospora hariotii Gäum. (1). Nevertheless, the pathogen on B. stachyoides has smaller sporangia as compared to those of P. hariotii (20 to 26 × 16 to 21 μm) (2) and it was closer to P. sordida (3). DNA of the pathogen from B. stachyoides was extracted and the cytochrome c oxidase subunit II (COX2) and internal transcribed spacer (ITS) regions were sequenced. Sequences were deposited in GenBank (COX2 as JX982637; ITS as JX982638). A BLAST search yielded 99% and 100% of maximum identity with P. sordida for COX2 and ITS, respectively. A more detailed phylogenetic study is necessary to clarify the relationship between P. sordida, P. hariotii, and related species causing downy mildew on closely related hosts. To our knowledge, this is the first report of P. sordida occurring on a member of the genus Buddleja. This is also the first time that P. sordida is reported from South America.

References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 19 October 2012. (2) S. M. Francis. C.M.I. Descr. Pathog. Fungi Bact. 767:1, 1983. (3) G. Hall. I.M.I. Descr. Fungi Bact. 1062:1, 1991. (4) K. G. Kissmann and D. Groth. Plantas Infestantes e Nocivas. São Paulo, BASF, 1997.
Intraoperative Infusion of 0.6–0.9 µg · kg−1 · min−1 Remifentanil Induces Acute Tolerance in Young Children after Laparoscopic Ureteroneocystostomy
Authors: Kim Sung-Hoon , Lee Min H. , Seo Hyungseok , Lee In-Gyu , Hong Jeong-Yeon , Hwang Jai-Hyun
Published: December 2012

Background:

Intraoperative infusion of opioids has been associated with increased postoperative pain and analgesic requirements, but the development of tolerance in young children is less clear. This prospective, randomized, double-blinded study was designed to test the hypothesis that the intraoperative administration of remifentanil results in postoperative opioid tolerance in a dose-related manner in young children.

Methods:

We enrolled 60 children (aged 1–5 yr) who were undergoing elective laparoscopic ureteroneocystostomy. Patients were randomized and received an intraoperative infusion of 0, 0.3, 0.6, or 0.9 µg·kg−1·min−1 remifentanil. Postoperative pain was managed by a parent/nurse-controlled analgesia pump using fentanyl. The primary outcome included the total fentanyl consumptions at 24 and 48 h postsurgery. Secondary outcomes were the postoperative pain scores and adverse effects.

Results:

The children who received 0.6 and 0.9 µg·kg−1·min−1 remifentanil required more postoperative fentanyl than the children who received saline or 0.3 µg·kg−1·min−1 remifentanil (all P < 0.001) for 24 h after surgery. The children who received 0.3–0.9 µg·kg−1·min−1 intraoperative remifentanil reported higher pain scores at 1 h after surgery than the children who received saline (P = 0.002, P = 0.023, and P = 0.006, respectively). No significant intergroup differences in recovery variables were observed, but vomiting was more frequent in the 0.9 µg·kg−1·min−1 remifentanil group than in the other groups (P = 0.027).

Conclusions:

The intraoperative use of 0.3 µg·kg−1·min−1 remifentanil for approximately 3 h (range: 140–265 min) did not induce acute tolerance, but the administration of 0.6 and 0.9 µg·kg−1·min−1 remifentanil to young children resulted in acute tolerance for 24 h after surgery in an apparently dose-related manner.
Parturition date for a given female is highly repeatable within five roe deer populations
Authors: Plard Floriane , Gaillard Jean-Michel , Bonenfant Christophe , Hewison A. J. Mark , Delorme Daniel , Cargnelutti Bruno , Kjellander Petter , Nilsen Erlend B. , Coulson Tim
Published: December 2012

Births are highly synchronized among females in many mammal populations in temperate areas. Although laying date for a given female is also repeatable within populations of birds, limited evidence suggests low repeatability of parturition date for individual females in mammals, and between-population variability in repeatability has never, to our knowledge, been assessed. We quantified the repeatability of parturition date for individual females in five populations of roe deer, which we found to vary between 0.54 and 0.93. Each year, some females gave birth consistently earlier in the year, whereas others gave birth consistently later. In addition, all females followed the same lifetime trajectory for parturition date, giving birth progressively earlier as they aged. Giving birth early should allow mothers to increase offspring survival, although few females managed to do so. The marked repeatability of parturition date in roe deer females is the highest ever reported for a mammal, suggesting low phenotypic plasticity in this trait.
Ecological change in the lower Omo Valley around 2.8 Ma
Authors: Bibi Faysal , Souron Antoine , Bocherens Hervé , Uno Kevin , Boisserie Jean-Renaud
Published: December 2012

Late Pliocene climate changes have long been implicated in environmental changes and mammalian evolution in Africa, but high-resolution examinations of the fossil and climatic records have been hampered by poor sampling. By using fossils from the well-dated Shungura Formation (lower Omo Valley, northern Turkana Basin, southern Ethiopia), we investigate palaeodietary changes in one bovid and in one suid lineage from 3 to 2 Ma using stable isotope analysis of tooth enamel. Results show unexpectedly large increases in C
4
dietary intake around 2.8 Ma in both the bovid and suid, and possibly in a previously reported hippopotamid species. Enamel
δ
13
C values after 2.8 Ma in the bovid (
Tragelaphus nakuae
) are higher than recorded for any living tragelaphin, and are not expected given its conservative dental morphology. A shift towards increased C
4
feeding at 2.8 Ma in the suid (
Kolpochoerus limnetes
) appears similarly decoupled from a well-documented record of dental evolution indicating gradual and progressive dietary change. The fact that two, perhaps three, disparate Pliocene herbivore lineages exhibit similar, and contemporaneous changes in dietary behaviour suggests a common environmental driver. Local and regional pollen, palaeosol and faunal records indicate increased aridity but no corresponding large and rapid expansion of grasslands in the Turkana Basin at 2.8 Ma. Our results provide new evidence supporting ecological change in the eastern African record around 2.8 Ma, but raise questions about the resolution at which different ecological proxies may be comparable, the correlation of vegetation and faunal change, and the interpretation of low
δ
13
C values in the African Pliocene.