bluuebunny/crossref_metadata_2025 huggingface dataset:
70 ONE-STEP CRYOPROTECTANT DILUTION FOLLOWING VITRIFICATION OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Morató R. , Mogas T.
Published: December 2012

Although slow freezing continues to be the most widely used technique of cryopreservation for bovine in vivo- and in vitro-produced embryos, vitrification has been tested in different species with good results, especially when dealing with in vitro-produced embryos. Vitrification represents a minor expense in time and equipment associated with cryopreservation compared with conventional slow freezing. However, vitrification, which is the most common method for human embryo cryopreservation, has not been widely adopted by embryo-transfer practitioners for commercial use in cattle. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting, and it is difficult to perform in the field. The objective of this study was to develop a one-step dilution method suitable for one-step bovine embryo transfer using the cryotop vitrification method. Embryos produced in vitro by standard procedures were vitrified at the blastocyst stage at Day 7 post-insemination in a mixture of 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose using cryotop devices. Embryos were randomly assigned to 1 of 3 warming methods: (1) W3: warming was carried out following the cryotop method (1 M sucrose for 1 min, 0.5 M sucrose for 3 min, and 0 M sucrose for 6 min); (2) W1/0.5: embryos were warmed directly in 0.5 M sucrose for 3 min; and (3) W1/0: embryos were warmed directly in 0 M sucrose for 5 min. Survival rates were assessed in terms of blastocyst re-expansion, hatching, and hatched status at 3 and 24 h after warming. Data were analyzed using the statistical analysis systems package (SAS, v9.1). Data from at least 3 replicates were collected. Comparisons of vitrified–warmed blastocyst survival rates between groups were performed using the chi-squared test. The level of statistical significance was set at P < 0.05. When embryo survival was evaluated at 3 h postwarming, embryos warmed using the 3-step dilution protocol and those warmed directly in 0.5 M sucrose showed higher percentages of survival (W3: 89.8%, n = 98; W1/0.5: 87.5%, n = 64; P < 0.05) than those blastocysts that were warmed directly in 0 M sucrose (W1/0: 66.4%, n = 146). However, similar rates irrespective of the warming procedure were observed at 24 h postwarming (W3: 85.7%, W1/0.5: 88.2%, W1/0: 70.5%). Warmed in vitro-produced embryos exposed to W3 (47.6%) and W1/0.5 (35.6%) achieved higher percentages of embryos developing to the hatched blastocyst stage after 24 h of culture than those embryos warmed in W1/0 (20.4%; P < 0.05). Our results indicate that direct warming and dilution of cyotop-vitrified embryos in 0.5 M sucrose for 3 min may enable one-step bovine embryo transfer without requirement of a microscope or other laboratory equipment, simplifying the embryo-transfer procedure of vitrified embryos on farm at the same level of complexity as carrying out AI.
Support came from Spanish MEC (RZ2010-00015-0-00; AGL2010-19069) and Generalitat de Catalunya (2009 SGR 621).
72 EFFECT OF SINGLE-LAYER CENTRIFUGATION WITH EQUIPURE™ ON MOTILITY KINEMATICS OF FROZEN - THAWED DONKEY SPERM
Authors: Ortiz I. , Dorado J. , Acha D. , Ramirez L. , Urbano M. , Galvez M. J. , Alcaraz L. , Portero J. M. , Quesada F. , Gonzalez C. , Demyda-Peyras S. , Hidalgo M.
Published: December 2012

Single-layer centrifugation (SLC) with EquipureTM Bottom Layer has been used to enhance the quality of stallion semen samples; however, no studies have been performed on donkeys. The aim of this study was to determine if SLC with EquipureTM Bottom Layer improves kinematic parameters on frozen–thawed donkey sperm. Semen was collected from 4 Andalusian donkeys by artificial vagina. Three ejaculates from each donkey were centrifuged with EquiproTM, supernatant was removed, and pellet was re-extended in the freezing medium GentTM to a final concentration of 200 × 106 spermatozoa per milliliter. Sperm were slowly cooled to 5°C for 2 h, loaded in 0.5-mL plastic straws, and frozen in liquid-nitrogen vapors. After at least one week of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: aliquot 1 was used as such (control) and aliquot 2 was processed by SLC using EquipureTM Bottom Layer. Computer-assisted sperm analysis was performed, and sperm kinematics total motility (%), progressive motility (%), curvilinear velocity (VCL; µm s–1), velocity straight line (VSL; µm s–1), velocity average path (VAP; µm s–1), linearity (LIN; %), straightness (STR; %), wobble (WOB; %), lateral head displacement (ALH; µm), and beat cross frequency (BCF; Hz) were statistically compared using GLM model between frozen–thawed semen samples processed or not with EquipureTM. Results were expressed as mean ± standard error. Significant differences (P < 0.05) were found between SLC-selected and unselected semen for total motility (77.44 ± 5.83 v. 58.89 ± 6.07), progressive motility (76.88 ± 4.52 v. 56.59 ± 5.44), VCL (137.50 ± 0.75 v. 133.0 ± 0.99), LIN (69.43 ± 0.31 v. 68.23 ± 0.41), STR (78.45 ± 0.29 v. 76.90 ± 0.37), WOB (85.06 ± 0.18 v. 83.91 ± 0.26), ALH (2.76 ± 0.01 v. 2.44 ± 0.01), and BCF (9.13 ± 0.05 v. 8.53 ± 0.06), respectively. No significant differences were observed for VSL (102.89 ± 0.70 v. 104.32 ± 0.95) and VAP (123.21 ± 0.71 v. 121.50 ± 0.98). Most of the computer-assisted sperm analysis parameters used in the present study have been previously identified as reliable markers of sperm motility in relation to sperm quality and fertility. It has also been reported that VCL appears to be critical for the formation of the sperm reservoir and penetration of the zona pellucida. In addition, other variables improved in the SLC-selected samples have been described as measure of progressivity (LIN, STR) and spermatozoa vigor (BCF, ALH). These preliminary results suggest an additional option for improving sperm quality in donkey semen doses. In conclusion, SLC with EquipureTM can be used to enhance kinematic parameters on frozen–thawed donkey sperm.
81 VITRIFICATION OF IMMATURE PORCINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED SOLUTION
Authors: Takahashi D. , Kuwayama M. , Funahashi H.
Published: December 2012

A high concentration of serum supplements has been contained in basic vitrification solutions to protect plasma membranes. The objective of this study was to examine if vitrification of immature porcine oocytes could be achieved successfully in a chemical-defined solution containing 0.6 mg mL–1 hydroxypropyl cellulose (HPC). Cumulus–oocyte complexes (COC) were aspirated from follicles 3 to 6 mm in diameter in abattoir-derived porcine ovaries. The COC or denuded oocytes were vitrified according to a commercial protocol of Cryotop (Kitazato BioPharma Co. Ltd., Shizuoka, Japan) with original solutions or modified ones that had serum supplement replaced with HPC. After vitrification and warming, viabilities of oocytes and cumulus cells were evaluated under a fluorescent microscope after staining with fluorescein diacetate and propidium iodide (Table 1). Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance, P < 0.05). Although viabilities of vitrified/warmed oocytes in all groups were significantly lower (82.7–89.1%) than those of fresh controls (99.5%), there were no significant differences among vitrified groups. When COC were vitrified, viability of cumulus cells (54.3%) in HPC group was not different from that of nonvitrified controls (72.5%) but higher than that in the original solution group (48.1%). In conclusion, these results demonstrate that HPC rather than serum supplement could be a suitable chemically defined supplement for vitrification of immature porcine COC.

Table 1.Viabilities of vitrified/warmed porcine oocytes and cumulus cells1
82 VIABILITY OF SHEEP-SKIN FIBROBLASTS AFTER SLOW FREEZING
Authors: Toishibekov Y. , Belyaev N. , Blackburn H. , Tleulieva R. , Katubayeva B. , Tursunova R. , Nurkenov T.
Published: December 2012

Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of different species is of paramount importance, because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of different cooling rates on viability of frozen–thawed sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors (Table 1). Skin samples collected from 10 adult sheep were cut on small pieces (1 × 1 mm), placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and covered with coverslips followed by incubation at 5% CO2, 95% relative humidity, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s phosphate buffered saline + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300g for 10 min. For cryoconservation, fibroblast samples were then diluted at a concentration of 2 × 106 cells mL–1 in DMEM + 20% FBS and 10% dimethyl sulfoxide or 10% ethylene glycol and placed into 0.25-mL plastic straws or 2-mL cryovials. Straws were sealed with modeling clay and maintained at +5°C for 120 min before freezing. Cryopreservation of fibroblasts was carried out by 2 procedures: (1) straws were frozen in programmable freezer Kryo Planer 360-3,5 using the following freezing regimen: +5°C to –40°C at –1°C min–1, –40°C to –85°C at –4°C min–1, and then plunged into liquid nitrogen; (2) cryovials were placed in a Styrofoam box and loaded into a freezer at –70°C for 24 h, and then samples were plunged into liquid nitrogen for storage. Samples were thawed for 1 min in a 37°C water bath. Frozen–thawed samples were diluted with DMEM (1 : 5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL–1. Viability of frozen–thawed fibroblast samples was detected using the Trypan Blue staining method. The values obtained (Table 1) are expressed as mean standard error of the mean (SEM). Statistical analysis was done using Student’s test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. However, there were no differences between the cooling procedures. Importantly, our data suggest that the use of 1.5-M ethylene glycol reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryoconservation.

Table 1.Effect of 2 cryoprotectant agent (CPA) on the viability of frozen–thawed ovine fibroblasts
84 EFFECT OF A STRESSOR ON CANINE SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST
Authors: Urbano M. , Dorado J. , Ortiz I. , Galvez M. J. , Demyda-Peyras S. , Moreno M. , Alcaraz L. , Ramirez L. , Quesada F. , Gonzalez C. , Portero J. M. , Acha D. , Hidalgo M.
Published: December 2012

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for human and different mammalian species (Sperm-Halomax®), based on the sperm chromatin dispersion test (SCDt); however, no studies has been performed specifically on canine frozen–thawed-stressed semen but is there for cooled semen. The aim of this work was to assess the effect of a stressor (24 h in an oven at 38°C) on canine frozen–thawed semen using the SCDt to resemble what happens in the female reproductive tract. For this purpose, ejaculates were collected by digital manipulation from 4 healthy beagle dogs and the sperm-rich fraction of the ejaculates from 3 different dogs was pooled each time. All the pooled semen samples (n = 4) used presented physiological values concerning to routine semen parameters (motility, morphology, and sperm concentration). After evaluation, semen samples were centrifuged and the sperm pellet resuspended to a final concentration of 100 × 106 sperm mL–1 in 2 steps with CaniPRO Freeze (Minitub, Tiefenbach, Germany). Sperm were slowly cooled to 5°C and then loaded into 0.5-mL plastic straws. After that, straws were frozen in liquid-nitrogen vapours for 10 min and stored into a nitrogen tank. Straws were thawed in a water bath (30 s/37°C) and incubated for 24 hours at 38°C before analysis. The sperm DNA fragmentation was assessed in fresh semen and frozen–thawed-stressed samples using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain) following the manufacturer’s instructions. Slides were stained for green fluorescence staining and 500 sperm per slide were counted using fluorescence microscopy. The sperm DNA fragmentation index (%) was compared between fresh and frozen–thawed-stressed semen samples by ANOVA. Results were expressed as mean ± standard error of the mean. The results obtained showed that subjecting thawed semen to 24 h in an oven at 38°C significantly increased (P < 0.05) DNA fragmentation compared with fresh semen (2.7% ± 0.2 v. 1.4 ± 0.1%). The stress factor was performed to simulate the viability of canine thawed sperm (12–24 h) when a bitch is inseminated with frozen semen. It would be interesting to perform further studies to relate sperm DNA fragmentation and fertility of frozen–thawed canine semen. In conclusion, frozen–thawed-stressed semen samples increased the sperm DNA fragmentation index measured using a SCDt. Further studies are needed to relate sperm DNA fragmentation with fertility rates or cryopreservation success.
Glycosylation facilitates transdermal transport of macromolecules
Authors: Pino Christopher J. , Gutterman Jordan U. , Vonwil Daniel , Mitragotri Samir , Shastri V. Prasad
Published: December 2012

Stratum corneum, the outermost layer of skin, allows transport of only low-molecular weight (<500) lipophilic solutes. Here, we report a surprising finding that avicins (
Av
s), a family of naturally occurring glycosylated triterpenes with a molecular weight > 2,000, exhibit skin permeabilities comparable to those of small hydrophobic molecules, such as estradiol. Systematic fragmentation of the
Av
molecule shows that deletion of the outer monoterpene results in a 62% reduction in permeability, suggesting an important role for this motif in skin permeation. Further removal of the tetrasaccharide residue results in a further reduction of permeability by 79%. These results, taken in sum, imply that synergistic effects involving both hydrophobic and hydrophilic residues may hold the key in facilitating translocation of
Av
s across skin lipids. In addition to exhibiting high permeability,
Av
s provided moderate enhancements of skin permeability of estradiol and polysaccharides, including dextran and inulin but not polyethylene glycol.
Extent and relevance of stacking disorder in “ice Ic”
Authors: Kuhs Werner F. , Sippel Christian , Falenty Andrzej , Hansen Thomas C.
Published: December 2012

A solid water phase commonly known as “cubic ice” or “ice Ic” is frequently encountered in various transitions between the solid, liquid, and gaseous phases of the water substance. It may form, e.g., by water freezing or vapor deposition in the Earth’s atmosphere or in extraterrestrial environments, and plays a central role in various cryopreservation techniques; its formation is observed over a wide temperature range from about 120 K up to the melting point of ice. There was multiple and compelling evidence in the past that this phase is not truly cubic but composed of disordered cubic and hexagonal stacking sequences. The complexity of the stacking disorder, however, appears to have been largely overlooked in most of the literature. By analyzing neutron diffraction data with our stacking-disorder model, we show that correlations between next-nearest layers are clearly developed, leading to marked deviations from a simple random stacking in almost all investigated cases. We follow the evolution of the stacking disorder as a function of time and temperature at conditions relevant to atmospheric processes; a continuous transformation toward normal hexagonal ice is observed. We establish a quantitative link between the crystallite size established by diffraction and electron microscopic images of the material; the crystallite size evolves from several nanometers into the micrometer range with progressive annealing. The crystallites are isometric with markedly rough surfaces parallel to the stacking direction, which has implications for atmospheric sciences.
Heterobivalent ligands target cell-surface receptor combinations in vivo
Authors: Xu Liping , Josan Jatinder S. , Vagner Josef , Caplan Michael R. , Hruby Victor J. , Mash Eugene A. , Lynch Ronald M. , Morse David L. , Gillies Robert J.
Published: December 2012

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.
Nonreceptor tyrosine phosphatase Shp2 promotes adipogenesis through inhibition of p38 MAP kinase
Authors: He Zhao , Zhu Helen H. , Bauler Timothy J. , Wang Jing , Ciaraldi Theodore , Alderson Nazilla , Li Shuangwei , Raquil Marie-Astrid , Ji Kaihong , Wang Shufen , Shao Jianhua , Henry Robert R. , King Philip D. , Feng Gen-Sheng
Published: December 2012

The molecular mechanism underlying adipogenesis and the physiological functions of adipose tissue are not fully understood. We describe here a unique mouse model of severe lipodystrophy. Ablation of
Ptpn11/Shp2
in adipocytes, mediated by
aP2-Cre
, led to premature death, lack of white fat, low blood pressure, compensatory erythrocytosis, and hepatic steatosis in
Shp2

fat−/−

mice. Fat transplantation partially rescued the lifespan and blood pressure in
Shp2

fat−/−

mice, and administration of leptin also restored partially the blood pressure of mutant animals with endogenous leptin deficiency. Consistently, homozygous deletion of Shp2 inhibited adipocyte differentiation from embryonic stem (ES) cells. Biochemical analyses suggest a Shp2-TAO2-p38-p300-PPARγ pathway in adipogenesis, in which Shp2 suppresses p38 activation, leading to stabilization of p300 and enhanced PPARγ expression. Inhibition of p38 restored adipocyte differentiation from
Shp2

−/−

ES cells, and p38 signaling is also suppressed in obese patients and obese animals. These results illustrate an essential role of adipose tissue in mammalian survival and physiology and also suggest a common signaling mechanism involved in adipogenesis and obesity development.
Decoding the rules of recruitment of excitatory interneurons in the adult zebrafish locomotor network
Authors: Ausborn Jessica , Mahmood Riyadh , El Manira Abdeljabbar
Published: December 2012

Significance

Spinal neural networks generate locomotion. An adjustment of the locomotion speed entails a precise order of recruitment of excitatory interneurons (e.g., V2a interneurons) within these networks. We show, using the adult zebrafish spinal cord, that the recruitment order of V2a interneurons is not topographic and does not conform to input resistance. The incremental recruitment of these interneurons is determined by scaling the excitatory drive with input resistance. We also show that locomotor networks are composed of multiple microcircuits recruited in a continuum. Thus, we provide insights into the recruitment mechanisms of spinal microcircuits that ensure optimal execution of locomotor movements.
p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs
Authors: Leonova Katerina I. , Brodsky Leonid , Lipchick Brittany , Pal Mahadeb , Novototskaya Liliya , Chenchik Alex A. , Sen Ganes C. , Komarova Elena A. , Gudkov Andrei V.
Published: December 2012

Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2’-deoxycytidine. The abundance of these transcripts exceeded the level of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named “TRAIN” (for “transcription of repeats activates interferon”), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.
Distinct mechanisms mediate naïve and memory CD8 T-cell tolerance
Authors: Jellison Evan R. , Turner Michael J. , Blair David A. , Lingenheld Elizabeth G. , Zu Li , Puddington Lynn , Lefrançois Leo
Published: December 2012

Peripheral tolerance to developmentally regulated antigens is necessary to sustain tissue homeostasis. We have now devised an inducible and reversible system that allows interrogation of T-cell tolerance induction in endogenous naïve and memory CD8 T cells. Our data show that peripheral CD8 T-cell tolerance can be preserved through two distinct mechanisms, antigen addiction leading to anergy for naïve T cells and ignorance for memory T cells. Induction of antigen in dendritic cells resulted in substantial expansion and maintenance of endogenous antigen-specific CD8 T cells. The self-reactive cells initially exhibited effector activity but eventually became unresponsive. Upon antigen removal, the antigen-specific population waned, resulting in development of a self-specific memory subset that recalled to subsequent challenge. In striking contrast to naïve CD8 T cells, preexisting antigen-specific memory CD8 T cells failed to expand after antigen induction and essentially ignored the antigen despite widespread expression by dendritic cells. The inclusion of inflammatory signals partially overcame memory CD8 T-cell ignorance of self-antigen. Thus, peripheral CD8 T-cell tolerance for naïve CD8 T cells depended on the continuous presence of antigen, whereas memory CD8 T cells were prohibited from autoreactivity in the absence of inflammation.
Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes
Authors: McCullough Shaun D. , Xu Xiaojiang , Dent Sharon Y. R. , Bekiranov Stefan , Roeder Robert G. , Grant Patrick A.
Published: December 2012

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder that results from polyglutamine expansion of the ataxin-7 (ATXN7) protein. Remarkably, although mutant ATXN7 is expressed throughout the body, pathology is restricted primarily to the cerebellum and retina. One major goal has been to identify factors that contribute to the tissue specificity of SCA7. Here we describe the development and use of a human astrocyte cell culture model to identify reelin, a factor intimately involved in the development and maintenance of Purkinje cells and the cerebellum as a whole, as an ATXN7 target gene. We found that polyglutamine expansion decreased ATXN7 occupancy, which correlated with increased levels of histone H2B monoubiquitination, at the reelin promoter. Treatment with trichostatin A, but not other histone deacetylase inhibitors, partially restored reelin transcription and promoted the accumulation of mutant ATXN7 into nuclear inclusions. Our findings suggest that reelin could be a previously unknown factor involved in the tissue specificity of SCA7 and that trichostatin A may ameliorate deleterious effects of the mutant ATXN7 protein by promoting its sequestration away from promoters into nuclear inclusions.
Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay
Authors: Alexandrov Andrei , Colognori David , Shu Mei-Di , Steitz Joan A.
Published: December 2012

The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the nonsense-mediated decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association. In vivo depletion of hCWC22, as for yeast Cwc22p, causes a splicing defect, resulting in decreased levels of mature cellular mRNAs. Nonetheless, hCWC22 depletion yields increased levels of spliced RNA from the unusual nonsense codon-containing U22 host gene, which is a natural substrate of NMD. To test whether hCWC22 acts in NMD through coupling splicing to EJC deposition, we searched for mutations in hCWC22 that affect eIF4AIII deposition without affecting splicing. Addition of hCWC22(G168Y) with a mutation at the putative hCWC22/eIF4AIII interface exacerbates the defect in splicing-dependent deposition of eIF4AIII(T334V) with a mutation reported to be in direct contact with mRNA, linking hCWC22 to the process of EJC deposition in vitro. Importantly, the addition of hCWC22(G168Y) affects deposition of eIF4AIII(T334V) without inhibiting splicing or the efficiency of deposition of the endogenous eF4AIII(WT) in the same reaction, demonstrating hCWC22’s specific role in eIF4AIII deposition in addition to its role in splicing. The essential splicing factor CWC22 has, therefore, acquired functions in EJC assembly and NMD during evolution from single-celled to complex eukaryotes.
Salt Stress Induces Accumulation of γ–Aminobutyric Acid in Germinated Foxtail Millet (Setaria italica L.)
Authors: Bai Qingyun , Yang Runqiang , Zhang Lixia , Gu Zhenxin
Published: December 2012

Effects of NaCl treatments on sprout length, the contents of soluble protein, free amino acids, and γ‐aminobutyric acid (GABA) and on glutamate decarboxylase (GAD) activity in germinated foxtail millet were investigated, and the regulating effects of exogenous Ca2+, along with lanthanum chloride (LaCl3, a specific inhibitor of the Ca2+ pathway) and ethylene glycol tetraacetic acid (EGTA, a chelator of Ca2+) under salt stress, on GABA accumulation in germinated millet were examined in this paper. The results showed that NaCl treatments caused a decrease in sprout length of millet. Low concentration of NaCl treatments increased soluble protein content, but high concentration decreased soluble protein content. The level of free amino acids, GAD activity, and GABA content increased significantly under NaCl stress. Exogenous Ca2+ application under NaCl stress further increased GAD activity and GABA content; the optimal concentration of Ca2+ for GAD activity and GABA accumulation was 5.0mM under 100mM NaCl for 48 hr, at which GABA content was 31.92 mg/100 g, a 1.41‐fold increase as compared with that in seeds under NaCl stress (22.64 mg/100 g). GAD activity and GABA accumulation in germinated millet decreased when treated with LaCl3 or EGTA under NaCl stress. Our results suggest that salt stress combined with Ca2+ treatment may be used for preparation of millet with higher GABA content, which can be used as a natural resource for functional foods.
A Survey of Phytophthora spp. in Midwest Nurseries, Greenhouses, and Landscapes
Authors: Leonberger A. J. , Speers C. , Ruhl G. , Creswell T. , Beckerman J. L.
Published: December 2012

A survey of nurseries, greenhouses, and landscapes was conducted from 2006 to 2008 in order to determine the prevalence and diversity of Phytophthora spp. From sites in Iowa, Michigan, Ohio, and, predominantly, Indiana, 121 Phytophthora isolates were obtained from 1,657 host samples spanning 32 host genera. Based on sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA, 11 Phytophthora spp. and two hybrid species were identified. A majority of the isolates were P. citricola (35.9%) or P. citrophthora (27.4%). Six isolates were confirmed as hybrids (four of P. cactorum × hedraiandra and two of P. nicotianae × cactorum) by cloning and sequencing the ITS region. Three P. cactorum × hedraiandra isolates were obtained from the same site, from three Rhododendron spp., which are known hosts to the parental species. The fourth isolate, however, was recovered out of a different location in a Dicentra sp., which is not a known host to either parental species, suggesting an expansion of host range of the hybrid isolate as compared with either parental species.
The Diversity of Strawberry latent ringspot virus in New Zealand
Authors: Tang Joe , Ward Lisa I. , Clover Gerard R. G.
Published: December 2012

Strawberry latent ringspot virus (SLRSV) is widespread in many countries, especially in Europe. The virus was thought to be uncommon in New Zealand, having only been recorded in Prunus spp. However, this study revealed that SLRSV infects a much wider range of hosts. From 1999 to 2009, SLRSV was isolated from anemone (Anemone × hybrida), blackberry (Rubus spp.), impatiens (Impatiens walleriana), pepino (Solanum muricatum), and tibouchina (Tibouchina sp.) in the North Island of New Zealand. These SLRSV isolates were identified using electron microscopy, mechanical inoculation, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction techniques. This is thought to be the first report of anemone, impatiens, pepino, and tibouchina as hosts of SLRSV. Phylogenetic analysis and host range suggest that the five newly identified New Zealand isolates belong to two distinct strains: blackberry and impatiens isolates represent one strain and the other three isolates, plus the flowering cherry isolate reported previously in New Zealand, represent another strain. Both these strains are distinct from isolates reported elsewhere in the world. The strain infecting blackberry and impatiens is especially different and produced an unusual reaction in mechanical inoculation tests on herbaceous indicators. It is postulated that SLRSV may have gone undetected on its wider host range in New Zealand due to the latent infection in some hosts. The relationship of SLRSV isolates between New Zealand and overseas and the transmission modes of this virus are also discussed.
Sudden Vegetation Dieback in Atlantic and Gulf Coast Salt Marshes
Authors: Elmer W. H. , Useman S. , Schneider R. W. , Marra R. E. , LaMondia J. A. , Mendelssohn I. A. , Jiménez-Gasco M. M. , Caruso F. L.
Published: December 2012

Salt marshes rank as the most productive ecosystems on the planet. Biomass production can be greater than 3 kg dry matter/m2/year, which is 40% more biomass than tropical rainforests produce. Salt marshes provide multiple benefits to mankind. For example, coastal communities receive protection from storm surges and wave erosion. Salt marshes absorb excess nitrogen and phosphorus from sewage and fertilizer run-off into rivers, which, in turn, prevents algal blooms and hypoxia in coastal waters. In addition, these unique ecosystems provide habitat and shelter for many hundreds of species of shellfish, finfish, migratory and sedentary birds, and other marine animals. Despite the richness in animal species, the intertidal marshes of the salt marsh ecosystem are dominated by only a few plant species. Of these, the most prevalent plant species in a marsh are the tall and short forms of smooth cordgrass (Spartina alterniflora). The first recorded account of a dieback in a U.S. salt marsh was in the early 1990s in the Florida panhandle where patches of Sp. alterniflora as large as 1 ha died. This article explores possible causes of Sudden Vegetation Dieback.
First Report of Leaf Spot Caused by Corynespora cassiicola on Baphicacanthus cusia in China
Authors: Li Q.-L. , Huang S.-P. , Guo T.-X. , Pan Z.-B. , Mo J.-Y. , Ning P. , Hsiang T.
Published: December 2012

Baphicacanthus cusia is a perennial herbaceous plant in the family Acanthaceae that is native to China, where it grows in warm temperate mountainous or hilly regions. It is commonly used as a Chinese herbal medicine. In March 2012, symptoms of leaf spot were observed on leaves of B. cusia in Long'an County, Guangxi, China, where this plant is extensively cultivated. Symptoms were initially small brown dots which developed into irregular to circular leaf spots. These spots enlarged and overlapped, extending until the 7- to 9-cm-long and 3- to 4-cm-wide leaves withered entirely, mostly within 2 months. On potato dextrose agar (PDA), the same fungus was cultured from 92% of 75 symptomatic leaf samples that had been surface sterilized in a 45-second dip in 0.1% mercuric chloride. Fungal structures were observed on diseased leaves: conidiophores (85 to 460 × 4 to 8 μm) were erect, brown, single or in clusters, and conidia (36 to 90 × 5 to 16 μm) were single or in chains of two to four, brown, cylindrical or obclavate, straight or slightly curved, with 3 to 18 pseudosepta and a conspicuous hilum. Three single-spore isolates were identified as Corynespora cassiicola (Berk & Curt.) Wei based on morphological and cultural characteristics (1). The rDNA internal transcribed spacer (ITS) region of one isolate, ZY-1, was sequenced (GenBank Accession No. JX908713), and it showed 100% identity to C. cassiicola, GenBank FJ852716, an isolate from Micronesia cultured from Ipomoea batatas (2). Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (5 × 104 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 60-day-old, 20-cm tall plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, dark brown spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of C. cassiicola from diseased leaves. To our knowledge, this is the first report of C. cassiicola infecting B. cusia worldwide.

References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009.
First Report of Lasiodiplodia theobromae Causing Dieback of Aquilaria sinensis in Taiwan
Authors: Fan M.-C. , Yeh H.-C. , Hong C.-F.
Published: December 2012

Incense trees (Aquilaria sinensis (Lour.) Gilg) belong to a plant family used for alternative medicine in China and the production of wood. In the summer of 2012, at a nursery in Niaosong district, Kaohsiung City, Taiwan, more than 30% of a total of 400 incense trees had dieback symptoms on twigs with leaves attached, leading to eventual death of the entire plant. Symptomatic twigs and trunk pieces from six trees were collected and discolored tissues were excised, surface sterilized in 0.5% sodium hypochlorite solution, rinsed in sterilized distilled water, dried on sterilized filter paper, and then placed in petri dishes containing 2% water agar (WA). The dishes were incubated at room temperature for 1 to 2 days to obtain fungal strains from diseased tissues. The hyphal tips from developing fungal colonies were transferred to potato dextrose agar (PDA, Difco) dishes and placed under UV light (12 h/day) at 30°C. The purified colonies were used as inoculum in the pathogenicity tests. Pathogenicity tests were performed on 2-month-old A. sinensis seedlings, each treatment had three plants. Each plant was wounded by removing bark of the twigs with a disinfected scalpel enough to place a mycelium plug (about 5 × 10 mm2) of 7-day-old fungal isolate on the wound. The inoculated area was wrapped with a wet paper towel and Parafilm. Control plants were treated with PDA plugs. The symptoms described above were observed on inoculated plants 4 to 8 days after inoculation whereas control plants did not show symptoms. Diseased twigs were cut and placed in a moist chamber 21 days after inoculation and conidia oozing from pycnidia were observed. The same fungal pathogen was reisolated from inoculated plants, but not from the control. To identify the pathogen, the fungus was cultured as described above. The colonies were initially white with green to gray aerial mycelium after 5 to 6 days and eventually turned darker. Immature conidia were hyaline and one-celled, but mature conidia were dark brown, two-celled, thin-walled, and oval-shaped with longitudinal striations. The average size of 100 conidia was 25.23 ± 1.97 × 13.09 ± 0.99 μm with a length/width ratio of 1.92. For the molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with primers ITS1 and ITS4 (2) and sequenced. The sequences were deposited in GenBank (Accession No. JX945583) and showed 99% identity to Lasiodiplodia theobromae (HM346871, GQ469929, and HQ315840). Hence, both morphological and molecular characteristics confirmed the pathogen as L. theobromae (Pat.) Griffon & Maubl (1). To the best of our knowledge, this is the first report of L. theobromae causing dieback on Incense tree. This disease threatens tree survival and may decrease the income of growers.

References: (1) W. H. Ko et al. Plant Dis. 88:1383, 2004. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.
Unravelling the determinants of insular body size shifts
Authors: McClain Craig R. , Durst Paul A. P. , Boyer Alison G. , Francis Clinton D.
Published: December 2012

The island rule, a pattern of size shifts on islands, is an oft-cited but little understood phenomenon of evolutionary biology. Here, we explore the evolutionary mechanisms behind the rule in 184 mammal species, testing climatic, ecological and phylogenetic hypotheses in a robust quantitative framework. Our findings confirm the importance of species’ ecological traits in determining both the strength and the direction of body size changes on islands. Although the island rule pattern appears relatively weak overall, we find strongest support for models incorporating trait, climatic and geographical factors in a phylogenetic context, lending support to the idea that the island rule is a complex phenomenon driven by interacting intrinsic and extrinsic mechanisms. Overall, we find that different clades may be evolutionarily predisposed to dwarfism or gigantism, but the magnitude of size changes depends more on adaptation to the novel island environment.
A gene regulatory motif that generates oscillatory or multiway switch outputs
Authors: Panovska-Griffiths Jasmina , Page Karen M. , Briscoe James
Published: December 2012

The pattern of gene expression in a developing tissue determines the spatial organization of cell type generation. We previously defined regulatory interactions between a set of transcription factors that specify the pattern of gene expression in progenitors of different neuronal subtypes of the vertebrate neural tube. These transcription factors form a circuit that acts as a multistate switch, patterning the tissue in response to a gradient of Sonic Hedgehog. Here, by simplifying aspects of the regulatory interactions, we found that the topology of the circuit allows either switch-like or oscillatory behaviour depending on parameter values. The qualitative dynamics appear to be controlled by a simpler sub-circuit, which we term the AC–DC motif. We argue that its topology provides a natural way to implement a multistate gene expression switch and we show that the circuit is readily extendable to produce more distinct stripes of gene expression. Our analysis also suggests that AC–DC motifs could be deployed in tissues patterned by oscillatory mechanisms, thus blurring the distinction between pattern-formation mechanisms relying on temporal oscillations or graded signals. Furthermore, during evolution, mechanisms of gradient interpretation might have arisen from oscillatory circuits, or vice versa.
Do first-time breeding females imprint on their own eggs?
Authors: Soler Manuel , Ruiz-Castellano Cristina , Carra Laura G. , Ontanilla Juan , Martín-Galvez David
Published: December 2012

The egg-recognition processes underlying egg rejection are assumed to be based on an imprinting-like process (a female learning the aspect of her own eggs during her first breeding attempt). The imprinting-like process and the misimprinting costs have been the objective of many theoretical models and frequently have a leading role in papers published on brood parasitism; however, an experiment has never been undertaken to test the existence of this imprinting-like process by manipulating egg appearance in first-time breeding females. Here, we present the first such experimental study using the house sparrow (
Passer domesticus
), which is a conspecific brood parasite and which has a good ability to reject conspecific eggs, as a model species. We found that contrary to what the hypothesis predicts first-time breeding females did not reject their own eggs in their second breeding attempt. This lack of response against unmanipulated eggs could indicate that females have an innate preference for their own eggs. However, in a second experimental group in which first-time breeding females were allowed to learn the aspect of their (unmanipulated) own eggs, none ejected manipulated eggs during their second clutch either—a finding that does not support the idea of recognition templates being inherited, but instead suggests that recognition templates could be acquired again at each new breeding attempt. Our results demonstrate that it is likely that egg discrimination is not influenced by egg appearance in the first breeding attempt.
Different chromatin interfaces of the Drosophila dosage compensation complex revealed by high-shear ChIP-seq
Authors: Straub Tobias , Zabel Angelika , Gilfillan Gregor D. , Feller Christian , Becker Peter B.
Published: December 2012

Transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila males is brought about by a ribonucleoprotein assembly called Male-Specific-Lethal or Dosage Compensation Complex (MSL-DCC). This machinery is formed in male flies and specifically associates with active genes on the X chromosome. After assembly at dedicated high-affinity “entry” sites (HAS) on the X chromosome, the complex distributes to the nearby active chromatin. High-resolution, genome-wide mapping of the MSL-DCC subunits by chromatin immunoprecipitation (ChIP) on oligonucleotide tiling arrays suggests a rather homogenous spreading of the intact complex onto transcribed chromatin. Coupling ChIP to deep sequencing (ChIP-seq) promises to map the chromosomal interactions of the DCC with improved resolution. We present ChIP-seq binding profiles for all complex subunits, including the first description of the RNA helicase MLE binding pattern. Exploiting the preferential representation of direct chromatin contacts upon high-energy shearing, we report a surprising functional and topological separation of MSL protein contacts at three classes of chromosomal binding sites. Furthermore, precise determination of DNA fragment lengths by paired-end ChIP-seq allows decrypting of the local complex architecture. Primary contacts of MSL-2 and MLE define HAS for the DCC. In contrast, association of the DCC with actively transcribed gene bodies is mediated by MSL-3 binding to nucleosomes. We identify robust MSL-1/MOF binding at a fraction of active promoters genome-wide. Correlation analyses suggest that this association reflects a function outside dosage compensation. Our comprehensive analysis provides a new level of information on different interaction modes of a multiprotein complex at distinct regions within the genome.
A Noninvasive Platform for Imaging and Quantifying Oil Storage in Submillimeter Tobacco Seed
Authors: Fuchs Johannes , Neuberger Thomas , Rolletschek Hardy , Schiebold Silke , Nguyen Thuy Ha , Borisjuk Nikolai , Börner Andreas , Melkus Gerd , Jakob Peter , Borisjuk Ljudmilla
Published: December 2012

While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 µm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.