bluuebunny/crossref_metadata_2025 huggingface dataset:
60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION
Authors: Lee S. , Khoirinaya C. , Jin J.-X. , Kim G. A. , Lee B.-C.
Published: December 2015

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development.
This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.
67 INHIBITION OF 5α-REDUCTASE DURING LATE GESTATION IN THE MARE
Authors: Wynn M. , Legacki E. , Conley A. , Loux S. , Esteller-Vico A. , Stanley S. , Squires E. , Troedsson M. , Ball B.
Published: December 2015

During the latter half of pregnancy in the mare, circulating concentrations of progesterone (P4) are low and a major bioactive progestogen, 5α-dihydroprogesterone (DHP), is present in high concentrations. DHP is formed through the activity of 5α-reductase, which converts P4 to DHP. Further metabolites of DHP have been attributed to fetal and myometrial quiescence. The purpose of this study was to examine the effects of a 5α-reductase inhibitor (dutasteride) on P4 metabolism and onset of parturition. Pregnant mares (n = 5) were treated with dutasteride (0.01 mg kg–1; IM), and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until foaling. Serum concentrations of P4 and DHP were determined with liquid chromatography/tandem mass spectrometry (LC/MS-MS) daily for 9 days preceding parturition. Endocrine data were analysed with a random effects mixed model with time, treatment (TRT), and time × TRT interaction. Gestational data were analysed with a Wilcoxon test. Although there was a significant effect of time on P4 and DHP, there was no effect of TRT or time × TRT on these progestogens. For the ratio of DHP/P4, there were significant effects of time, TRT, and time × TRT interaction such that the ratio of DHP/P4 was greater in the control than dutasteride-treated mares. Birth weight and gestational length were not different (P > 0.2) between the dutasteride-treated and control mares, although placental weights were greater (P < 0.05) in dutasteride-treated mares. Because the ratio of DHP/P4 was suppressed in dutasteride-treated mares, it appears that dutasteride was active in late gestational mares. Although gestational length and neonatal weights were not different between groups, placentas from dutasteride-treated mares were heavier than those from control mares. The reason for the increase in placental weights was not determined.
76 EFFECTS OF REDUCED ENVIRONMENTAL LIGHT ON THE IN VITRO MATURATION OF PIG OOCYTES
Authors: Parra-Forero L. Y. , Góngora A. , Romo-García S. , López Damian E. P. , Mendoza G. D. , Guevara J. A. , García-Contreras A.
Published: December 2015

The effects of light on the steps of embryo manipulation have been described in several species, including humans. There are reports in which exposure of these cells to UV rays from sunlight affects them epigenetically, which could lead to meiotic arrest that would prevent normal maturation from the germinal vesicle (GV) to metaphase II (MII) stage, which would compromise subsequent embryo viability. Development to the blastocyst was not evaluated. The objective of the study was to observe the difference in maturation capacity (GV to MII) of prepubertal gilt oocytes under conditions of reduced ambient light. Thirty Duroc ovaries were recovered at slaughter and immersed in saline (0.9% NaCl) supplemented with penicillin-G (100 IU mL–1) and streptomycin sulfate (100 mg mL–1) at 29 to 34°C for transport to the laboratory where they were punctured with an 18-gauge needle attached to a 10-mL syringe. GV oocytes were selected with at least 3 layers of compact cumulus cells and were incubated with TCM-199 for 42 h in total with replacement with fresh maturation medium at 22 h. Oocytes were subsequently denuded using 0.1% hyaluronidase, fixed with 2% formaldehyde, and stained with Hoechst 33342 (10 min). Thereafter, oocytes were evaluated under a fluorescence microscope for the stage of meiosis: GV, metaphase I (MI), anaphase I/telophase I (ATI), and MII. GV oocytes were divided into two groups of 60 each as follows: Group 1 = handling in ambient light (1 change of culture medium and evaluation at the stereoscope); Group 2 = handling with reduced light (the change of medium was carried out in a dark room with red light on the stereoscope. Chi-square and Student’s t-test with Minitab 16.0 statistical program were used, and differences were considered significant at P < 0.05. There were significant differences in the percentages of maturation stages between Group 1 and Group 2, respectively: GV = 3.33, 3.33% (P = 0.45); MI = 20, 13.3% (P = 0.037); ATI = 33.3, 26.6% (P = 0.03); and MII = 43.3, 56.6% (P = 0.019). In conclusion, rate of maturation to MII was significantly higher with decreased light exposure. There was no difference in the GV groups probably because there was no manipulation at this stage. Further studies are necessary to determine the amount of light needed to optimize oocyte viability and to assess any potential effects on blastocyst production.
80 REACTIVE OXYGEN SPECIES LEVEL IN PIG EMBRYOS CULTURED IN PRESENCE OF HYALURONAN
Authors: Gajda B. , Kucia M. , Smorag Z. , Romek M.
Published: December 2015

It has been reported that during in vitro embryo culture reactive oxygen species (ROS) are generated and are detrimental to embryo development. A recent study (Smorag et al., Proc. 9th ICPR, 2013, 110) demonstrated that an addition of 1 mg mL–1 of hyaluronan (HA) to porcine embryo culture medium improves the development of zygote to blastocyst stage and the quality of produced embryos. Moreover, the embryos cultured with HA showed lower inner mitochondrial membrane potential (Romek et al., 2015 Proc. Symp. Progress in Cell Biology: Mitochondria and Chloroplast, Krakow, 31). Based on the beneficial effect provided by supplementation of HA during embryo culture, we investigated the ROS level in porcine embryos cultured with HA. Porcine zygotes were obtained surgically after flushing the oviducts of superovulated and inseminated gilts. In the experimental group, zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 1 mg mL–1 of HA (CROMA, Pharma GmbH, Leobendorf, Austria), in an atmosphere containing 5% CO2 in air, at 39°C. In the control group, HA supplementation was omitted. To measure ROS level, embryos at the stages 2–4 and 8–16 cell, morula, and blastocyst (experimental group) and zygote, 2–4 and 8–16 cell, morula, and blastocyst (control group) were labelled with 5 μM CM-H2DCFDA dye (Molecular Probes Inc., OR, USA) for 30 min at 39°C. Labelled embryos were then examined under a Nikon Eclipse microscope with a CCD camera. The total amount of fluorescence emitted from each individual embryos and proportional to the ROS level was measured in arbitrary units. The data were analysed using one-way ANOVA and post-hoc Tukey test. ROS level (mean ± standard error of the mean) in the experimental group was 8.21 ± 2.65 (n = 25), 10.31 ± 3.13 (n = 18), 9.08 ± 2.89 (n = 21), and 20.45 ± 2.38 (n = 31) for 2–4 cell, 8–16 cell, morula, and blastocyst, respectively, whereas in the control group was 9.15 ± 3.43 (n = 15), 7.11 ± 3.13 (n = 18), 8.67 ± 3.04 (n = 19), 11.47 ± 2.46 (n = 29), and 54.74 ± 2.89 (n = 21) for zygote, 2–4 cell, 8–16 cell, morula, and blastocyst, respectively. For experimental and control groups, ROS levels remained unchanged up to morula. On the contrary, at the blastocyst stage from the experimental group ROS level decreased significantly (P ≤ 0.05) in comparison with blastocysts from the control group. In conclusion, porcine blastocysts derived from zygotes cultured with supplementation of 1 mg of HA possess a significantly lower ROS level than blastocysts cultured without HA. This suggests that HA supplementation in culture medium can reduce the ROS level in porcine cultured blastocysts.
The project was funded by the National Science Center based on decision number DEC-2012/07/B/NZ9/01326.
87 COAGULANSIN-A VIA HEAT SHOCK PROTEIN 70 INDUCTION SHOWS BENEFICIAL EFFECTS ON THE DEVELOPMENT OF BOVINE EMBRYOS IN VITRO
Authors: Khan I. , Lee K.-L. , Ha A.-N. , Park P.-R. , Song S.-H. , Chowdury M. M. R. , Xu L. , Joo M.-D. , Kong I.-K.
Published: December 2015

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans, which belong to Solanaceae family. The coagulansin-A induces heat shock protein 70 (HSP-70), which acts as a cellular antioxidant. This study was conducted to investigate the effect of coagulansin-A on bovine oocytes maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To analyse the possible beneficial effect of coagulansin-A on bovine oocytes maturation in vitro, 355 oocytes per group (control and treatment) in seven replicates were subjected with three concentrations i.e. (1, 5, and 10 µM) of coagulansin-A. The coagulansin-A was added in the in vitro-matured (IVM) media for 20 to 22 h followed by IVF for 18 to 22 h, and after fertilization the fertilized oocytes were transferred to IVC1 media for 3 days. After 3 days, the cleavage rate was checked and the 8-cell stage embryos were transferred to IVC2 media and embryo development was checked at Day 8. The culture was carried out at 5% CO2 and 38.5°C. The results indicated that among the three concentrations of Coagulansin-A, only 5 µM remarkably (P < 0.05) improved embryo development (Day 8 blastocyst), being 27.30% and 40.01% for control and treated groups, respectively. This concentration also significantly (P < 0.05) encouraged the activation of HSP-70, having 16.44 arbitrary units (AU) and 35.41 AU integral optical density (IOD) for control and treated groups, respectively. The immunofluorescence analysis revealed that 5 µM coagulansin-A supplementation significantly (P < 0.05) inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing IOD of 8-Oxoguanosine (8-OxoG) from 28.12 AU in control to 18.06 AU for the treated group and nuclear factor kappa B (NF-kB) IOD (P < 0.05) from 42.25 AU to 21.80 for control and treated groups, respectively. Additionally, the results obtained from terminal deoxynucleotidyl transferase TUNEL assay confirmed that coagulansin-A treatment reduced the bovine embryo DNA damage significantly (P < 0.05) from 7.4 ± 0.375 to 5.7 ± 0.287 and improved the embryo quality (P < 0.05) with mean cell numbers of 127.7 ± 4.161 and 150.1 ± 3.624 per embryo for control and coagulansin-A treated groups, respectively. This study provides new information regarding the mechanisms by which coagulansin-A promotes bovine oocyte maturation and embryo development in vitro.
89 EFFECT OF MEDIA, METABOLIC REGULATORS, AND STAGE OF DEVELOPMENT ON LIPID CONTENT AND MITOCHONDRIAL POLARITY OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS
Authors: Roberts M. A. , Campos-Chillon L. F. , Barceló-Fimbres M. , Altermatt J. L.
Published: December 2015

Current bovine embryo culture methods result in accumulation of lipids and reactive oxygen species, possibly due to sub-optimal metabolic regulation. These effects decrease the cryopreservation survival and implantation potential of in vitro-produced (IVP) embryos. Forskolin has been shown to decrease lipid accumulation, and vitamin K2 (Vit K2) is thought to decrease oxidative stress from in vitro conditions. The aims of this study were (1) to assess lipid content of embryos cultured with or without forskolin and Vit K2 in both continuous and sequential SOF-based medium, and (2) to examine individual and combined effects of forskolin and Vit K2 on mitochondrial polarity. For Experiment 1, a 2 × 2 × 2 factorial design was used to compare culture systems (continuous v. 3-step sequential), additives (no additive v. Vit K2 (0.5 mM at Day 3) plus forskolin (10 µM at Day 5), and blastocyst stage [6 (early) v. 7 (late)] on overall lipid content. For Experiment 2, mitochondrial polarity of stage 7 blastocysts was analysed from the following groups: no additive, Vit K2 (0.5 mM at Day 3), forskolin (10 µM at Day 5), and Vit K2 plus forskolin. IVP embryos (n = 199, Experiment 1; n = 45, Experiment 2) were produced by standard procedures and cultured at 38.5°C in 5% O2, 5% CO2, and 90% N2. For Experiment 1, embryos were stained with 1 μg mL–1 Nile Red, and two images per embryo were taken along the equatorial plane at 40× magnification. For Experiment 2, embryos were stained with 300 nM MitoTracker Red CMX-Rosamine, and 10 images per embryo were acquired by confocal microscopy with a 5-μm step size at 40× magnification. For both experiments, fluorescence intensity (FI) of each image was measured by Image PRO software with embryo controlled for and background fluorescence corrected. Data (Table 1) were analysed by ANOVA and means were compared by Tukey’s HSD. In Experiment 1, embryos cultured with forskolin and Vit K2 showed decreased lipid content in both the early and late stage (P < 0.05), with no effect from culture system (P > 0.05). In Experiment 2, forskolin and Vit K2 individually increased mitochondrial polarity (P < 0.05), but had no combined effect (P > 0.05). In conclusion, these data suggest that while a combination of forskolin and Vit K2 as media additives reduces lipid accumulation, the interaction between these metabolic regulators may negate their individual effects on mitochondrial polarity.

Table 1.Fluorescence intensity of Nile Red and MitoTracker Red dyes between treatment groups
92 INSULIN-LIKE GROWTH FACTOR-1 PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS PRODUCED IN VITRO FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE
Authors: Rocha-Frigoni N. A. S. , Leão B. C. S. , Dall'Acqua P. C. , Ambrogi M. , Mingoti G. Z.
Published: December 2015

The objective of this study was to evaluate the protective effect of insulin-like growth factor (IGF-1) on blastocyst development and cryotolerance of bovine embryos in in vitro culture (IVC) under oxidative stress induced by menadione (MD). Cumulus-oocyte complexes (n = 1421) were matured in TCM-199 with bicarbonate, hormones, and 10% FCS for 22 h. After fertilization, the presumptive zygotes were cultured up to 7 days in SOF medium with 2.5% FCS and 0.5% BSA (control), and also supplemented with 100 μM IGF-1 (IGF). At Day 6, MD was included in the culture medium (0 μM, control; or 5.0 μM, MD) during 24 h. Cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7 (IVF = Day 0). At Day 7, a sample of the blastocysts was stained with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the intracellular ROS levels or was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA). Stained embryos were immediately evaluated under an epifluorescence microscope (excitation 495/550 nm and emission 404/590 nm, respectively, for ROS and TUNEL), and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. Other blastocysts were vitrified (Ingámed®, Maringá-PR, Brazil), and after warming, they were cultured for 24 h to evaluate the re-expansion rates. The results were compared by ANOVA followed by Student’s t-test (mean ± s.e.M) and re-expansion rates by chi-square test (P < 0.05). The cleavage rates did not differ (P > 0.05) among groups (77.1 ± 1.9% to 82.75 ± 2.2%). The blastocyst rates were similar between control (35.4 ± 2.0%) and IGF (34.5 ± 3.7%), and both were higher (P < 0.05) than MD (21.3 ± 2.7%); the IGF+MD group (28.3 ± 1.6%) was similar (P > 0.05) to all groups. The intracellular levels of ROS were higher (P < 0.05) for the MD group (21.7 ± 0.7) than for control (17.0 ± 1.6), and both were similar (P > 0.05) to the IGF (19.2 ± 0.6) and IGF+MD (18.0 ± 1.0) groups. The highest rates of apoptosis were found in the MD group (22.3% ± 2.3) and the smallest in IGF (9.1% ± 0.7), and both differed (P < 0.05) from control (12.8% ± 1.0), and IGF+MD (15.6% ± 1.6). The re-expansion rates were similar between control (77.4%) and IGF (69.2%), and both were higher (P < 0.05) than MD (49.1%); however, the IGF+MD group (57.6%) was similar (P > 0.05) to IGF and MD groups. In conclusion, the supplementation with IGF-1 during IVC reversed the detrimental effects of MD on embryonic levels of ROS and apoptosis, as well as improved the embryo development and cryotolerance of blastocysts under oxidative stress.
Financial support was provided by FAPESP (#2012/10083–8 and #2013/07382–6).
93 DEVELOPMENT OF BOVINE PRE-IMPLANTATION EMBRYOS IS ALTERED BY ADDITION OF ACTIVIN AND CONNECTIVE TISSUE GROWTH FACTOR FROM DAYS 5 TO 7 AFTER FERTILIZATION
Authors: Kannampuzha-Francis J. , Hansen P. J.
Published: December 2015

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. Here we tested actions of two molecules that are highly expressed in the endometrium for actions to modify development of bovine embryos. The molecules tested were activin and connective tissue growth factor (CTGF), and endpoints were percentage of fertilized oocytes becoming blastocysts and number of inner cell mass (ICM) and trophectoderm (TE) cells in Day 7 blastocysts. Bovine embryos produced in vitro from slaughterhouse ovaries were cultured in a serum-free culture medium. On Day 5 of culture, culture drops were supplemented with vehicle (control), human recombinant activin or CTGF (10–11, 10–10, or 10–9 M). On Day 7, blastocysts (n = 202) were collected and labelled with a nuclear dye (Hoescht 33342) and a TE cell marker (anti-CDX2). Statistical analysis was performed using the GLM procedure of SAS. Results are shown as least squares means ± s.e.M. The percentage of putative zygotes becoming a blastocyst on Day 7 was 28.4 ± 1.5% for control embryos; 32.2 ± 1.5%, 30.7 ± 1.5%, and 33.1 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M activin, respectively; and 27.8 ± 1.5%, 25.1 ± 1.5%, and 28.4 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M CTGF, respectively. Activin increased the proportion of putative zygotes becoming a blastocyst when added at 10–9 M (P = 0.0321) and tended to have the same effect at 10–11 M (P = 0.0898). There was no effect of any concentration of CTGF on development of putative zygotes to blastocyst on Day 7. There was no effect of treatment on total cell number or number of TE or of activin on ICM cell number or TE : ICM ratio. However, CTGF increased (P = 0.0011) the number of ICM (40.1 ± 1.5, 46.2 ± 1.4, 46.5 ± 1.4, and 56.3 ± 1.4 for control and 10–11, 10–10, and 10–9 M CTGF, respectively). CTGF also reduced (P = 0.0319) the TE : ICM ratio. Values were 2.8 ± 0.2 for control and 2.1 ± 0.2, 1.9 ± 0.2, and 1.6 ± 0.2 for 10–11, 10–10, and 10–9 M CTGF, respectively. Results indicate that activin and CTGF can affect embryo development. In particular, activin increases competence of embryos to develop to the blastocyst stage and CTGF affects blastocyst differentiation.
Support was provided by NIH R03 HD080855.
Design of a single protein that spans the entire 2-V range of physiological redox potentials
Authors: Hosseinzadeh Parisa , Marshall Nicholas M. , Chacón Kelly N. , Yu Yang , Nilges Mark J. , New Siu Yee , Tashkov Stoyan A. , Blackburn Ninian J. , Lu Yi
Published: December 2015

Significance

Nature spent millions of years to evolve electron transfer proteins that span a wide range of reduction potentials (E°′) under physiological conditions, from −1 V to 1 V vs. standard hydrogen electrode. Understanding the rules that govern such tuning within similar classes of metalloproteins enables scientists to rationally tune the E°′of their catalysts without changing the active site. An ultimate test of such understanding is to achieve the entire range of E°′ within a single protein, a feat that has not been achieved yet. Herein, we conclusively found that we can cover the entire 2-V range of E°′ using a single protein with five mutations and two metal ions. We have also provided explanations for structural features responsible for such high potential.
Conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing
Authors: Chalamcharla Venkata R. , Folco H. Diego , Dhakshnamoorthy Jothy , Grewal Shiv I. S.
Published: December 2015

Significance

RNA-mediated heterochromatin assembly requires transcription by RNA polymerases and a plethora of RNA-processing factors. However, the mechanisms that differentiate productive gene transcription from transcriptional activity linked to the assembly of repressive heterochromatin have remained elusive. Here we find that factors promoting noncanonical termination of RNA polymerase II transcription trigger heterochromatin assembly. We propose that termination and 3′-end processing factors serve as molecular sensors that, in addition to determining the fate of target transcripts, mediate heterochromatin assembly. Importantly, these findings provide insights into the paradoxical interdependence of heterochromatin assembly and transcription of target loci and suggest a mechanism for specifying certain genomic regions, including developmental genes and repeat elements, as targets for heterochromatin assembly.
Cuspidal representations of reductive p-adic groups are relatively injective and projective
Authors: Meyer Ralf
Published: December 2015

Cuspidal representations of a reductive

p
p

-adic group 

G
G

over a field of characteristic different from 

p
p

are relatively injective and projective with respect to extensions that split by a

U
U

-equivariant linear map for any subgroup 

U
U

that is compact modulo the centre. The category of smooth representations over a field whose characteristic does not divide the pro-order of 

G
G

is the product of the subcategories of cuspidal representations and of subrepresentations of direct sums of parabolically induced representations.
A role of the sphingosine-1-phosphate (S1P)–S1P receptor 2 pathway in epithelial defense against cancer (EDAC)
Authors: Yamamoto Sayaka , Yako Yuta , Fujioka Yoichiro , Kajita Mihoko , Kameyama Takeshi , Kon Shunsuke , Ishikawa Susumu , Ohba Yusuke , Ohno Yusuke , Kihara Akio , Fujita Yasuyuki
Published: December 2015

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)–S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.
Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 inChlamydomonasflagella
Authors: Harris J. Aaron , Liu Yi , Yang Pinfen , Kner Peter , Lechtreck Karl F.
Published: December 2015

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.
The Effects of Anesthetic Technique on Postoperative Opioid Consumption in Ankle Fracture Surgery
Authors: Christensen Kristian P. , Møller Ann M. , Nielsen Jesper K. , Klausen Tobias W. , Sort Rune
Published: December 2015

Objectives:

To investigate the impact of common anesthetic techniques on postoperative opioid consumption in ankle fracture surgery.

Materials and Methods:

We performed a retrospective cohort study on 622 patients with isolated ankle fractures undergoing primary reconstructive surgery. Patients were identified by consecutive screening in the years 2008 to 2011. Our primary endpoint was the difference in the 24-hour postoperative opioid consumption after general anesthesia (GA) compared with different regional anesthesia modalities. Secondary endpoints were length of stay in the postanesthesia care unit, risk of postoperative nausea and vomiting, and postoperative length of hospital stay.

Results:

Opioid consumption in 4 anesthesia groups (equipotent doses of IV morphine) was as follows: GA: 21.9 mg (95% confidence interval [CI]±1.2), GA+peripheral nerve block (PNB): 16.3 mg (95% CI±1.1), spinal anesthesia (SA): 17.1 mg (95% CI±0.9), and SA+PNB: 14.6 mg (95% CI±1.0). We found significant differences between “GA” and all other groups (P≤0.01). The most pronounced difference is seen when GA is compared with SA+PNB, with a 33.4% opioid reduction in the latter group.

Discussion:

Regional anesthesia modalities reduce postoperative opioid consumption in ankle fracture surgery in comparison with GA. A benefit of PNBs is possibly due to an improved pain profile. Our study is retrospective and cannot predict the exact magnitude of this benefit.
The Surgical Management of Osteochondritis Dissecans of the Knee in the Skeletally Immature: A Survey of the Pediatric Orthopaedic Society of North America (POSNA) Membership
Authors: Yellin Joseph L. , Gans Itai , Carey James L. , Shea Kevin G. , Ganley Theodore J.
Published: December 2015

Background:

While the characteristics of osteochondritis dissecans (OCD) of the knee that require surgery to heal have been described, several surgical techniques/procedures exist with no consensus established regarding timing of treatment and specific surgical intervention. In this study, we aim to determine current trends in surgical treatment for OCD lesions in the skeletally immature who have failed 6 months of nonoperative management by surveying a large cohort of orthopaedic surgeons.

Methods:

An electronic survey designed using REDCap to capture surgeon treatment preferences for OCD lesions was distributed to members of the Pediatric Orthopaedic Society of North America (POSNA). The survey inquired about treating physicians’ training and demographics. It then offered a series of clinical vignettes alongside imaging describing patients with varying degrees of severity of OCD following nonoperative treatment. Surgeons were prompted to select from a variety of multiple-choice–based options for further patient management. Standard descriptive statistics were used to summarize and compare the responses.

Results:

Of the 129 POSNA members completing the pediatric survey, 97.7% were attending level orthopaedic surgeons, the majority identifying with an academic institution and treating mostly skeletally immature patients. In the skeletally immature population, the majority would treat intact, stable OCD lesions with drilling in a retroarticular or transarticular manner. Preferred treatment for unstable, salvageable lesions was screw fixation using bioabsorble materials or metal with variable pitch with no bone graft. The majority of respondents would treat unstable, unsalvageable OCD lesions with chondroplasty and osteochondral transplant/transfer or microfracture/drilling.

Conclusions:

The POSNA membership appears to agree on principle in terms of treatment modalities for various stages of OCD lesions in the skeletally immature, whereas individual techniques of achieving these principles may vary. Members endorse drilling for stable intact lesions; fixation for unstable, salvageable lesions; and “defect fill” for unsalvageable lesions.

Clinical Significance:

OCD surgical treatment patterns can be used in future studies to determine which techniques are most effective for given indications, with the goal of designing a research-proven optimal treatment regimen for skeletally immature patients.
Esophageal Perforation Caused by a Posterior Pedicle Screw: A Case Report
Authors: Sariyilmaz Kerim , Ozkunt Okan , Sungur Mustafa , Dikici Fatih , Domanic Unsal
Published: December 2015

Background:

Posterior spinal instrumented fusion has received widespread acceptance in adolescent idiopathic scoliosis (AIS) treatment, there have been some potential complications resulted from screw misplacement. Esophagus is one of the vital structures close to the upper thoracic vertebras and is at risk for potential injury.

Methods:

A 15-year-old girl who underwent posterior instrumented fusion for AIS was admitted to our department 10 years later with a complaint of dysphagia due to an esophageal perforation from a malpositioned T4 pedicle screw (PS). After implant removal and nasogastric tube placement, oral feeding was started after 3 days and the patient was discharged from hospital after 1 week.

Results:

In 12 weeks, the esophageal perforation healed without complications.

Conclusions:

This is the first case report of esophageal perforation by a posterior PS in an AIS patient. Esophagus is close to the upper thoracic vertebras and an anterior cortical perforation can cause esophageal injury if longer than 35 mm PSs are used. This unusual but potential complication must be kept in mind in scoliosis surgery.

Level of Evidence:

Level IV—case report.
High-resolution microendoscopy: a point-of-care diagnostic for cervical dysplasia in low-resource settings
Authors: Grant Benjamin D. , Fregnani José H.T.G. , Possati Resende Júlio C. , Scapulatempo-Neto Cristovam , Matsushita Graziela M. , Mauad Edmundo C. , Quang Timothy , Stoler Mark H. , Castle Philip E. , Schmeler Kathleen M. , Richards-Kortum Rebecca R.
Published: December 2015

Cervical cancer is the third leading cause of cancer-related death among women in low-to-middle income countries. Pap testing and pathological services are difficult to implement under these settings. Alternative techniques for the diagnosis of cervical precancer in these settings are needed to reduce the burden of the disease. The objective of this study was to evaluate the diagnostic accuracy of a low-cost, high-resolution microendoscope imaging system in identifying precancerous lesions of the cervix in vivo. A retrospective study of 59 patients undergoing colposcopy for an abnormal Pap test was performed at Hospital de Câncer de Barretos in Brazil. All patients underwent colposcopy as per standard of care, and acetowhite lesions were recorded. High-resolution microendoscopy (HRME) images were obtained from one colposcopically normal region and from all lesions observed on colposcopy. Biopsies of abnormal areas were obtained and reviewed by three independent, blinded pathologists and compared with HRME findings. The mean nuclear area and the median nuclear eccentricity were calculated from HRME images acquired from each site. A diagnostic algorithm to distinguish histopathologically diagnosed cervical intraepithelial neoplasias of grade 2 or more severe lesions (high grade) from less severe lesions (low grade) was developed using these parameters. A test of trend was used to analyze the relationship between HRME positivity and severity of histopathogical diagnosis. Fisher’s exact test was used to analyze differences in HRME positivity between high-grade and low-grade lesions. Evaluable images were obtained from 108 of 143 discrete sites. Of these, 71 sites were colposcopically normal or low grade according to histopathology and 37 were diagnosed as high grade on the basis of histopathology. Using the mean nuclear area and the median nuclear eccentricity, HRME images from 59 colposcopically abnormal sites were classified as high grade or low grade with 92% sensitivity and 77% specificity compared with histopathological findings. Increasing HRME positivity showed a significant trend with increasing severity of diagnosis (P
trend<0.001). We found a strong association (P<0.001) between HRME positivity and a histopathological diagnosis of cervical intraepithelial neoplasia of grade 2 or higher. HRME demonstrated an accurate in-situ diagnosis of high-grade dysplasia. In low-resource settings in which colposcopy and histopathology services are severely limited or unavailable, HRME may provide a low-cost, accurate method for diagnosis of cervical precancer without the need for biopsy, allowing for a single ‘screen-and-treat’ approach.
Comparison of Oncology Patients’ and Their Family Caregivers’ Attitudes and Concerns Toward Pain and Pain Management
Authors: Valeberg Berit Taraldsen , Miaskowski Christine , Paul Steven M. , Rustøen Tone
Published: December 2015

Background:

Both cancer patients and their family caregivers (FCs) report concerns about pain and pain management. When dyads share appraisal of the illness context, they may experience better dyadic adjustment.

Objective:

The aim of this study was to compare oncology outpatients’ and their FCs’ attitudes and concerns toward pain and pain management.

Methods:

In a cross-sectional study, outpatients with pain (n = 71) and their FCs completed the Barriers Questionnaire II, the Brief Pain Inventory, and information about demographic characteristics. Correlations and paired-samples t tests were calculated to evaluate agreement and differences in barrier scores between the patients and their FCs.

Results:

Congruence was found in patients’ and FCs’ beliefs about pain and the use of analgesics on 4 of 7 subscales (ie, tolerance, immune system, side effects, distract the medical doctor) and on the Barriers Questionnaire II total score. Both patients and their FCs were most concerned about addiction.

Conclusions:

The concerns that both patients and their FCs have about pain and the use of analgesics may act as barriers to effective pain management. Fear of addiction may be an important barrier to cancer pain management. Only small differences were found in concerns between the patients and their FCs.

Implications for Practice:

Patients and FCs need education about perceived barriers to effective pain management. They should be coached together to maintain or increase the congruence between them.
Healthcare Professionals’ Knowledge of Family Psychosocial Problems in Pediatric Cancer
Authors: Barrera Maru , Rokeach Alan , Yogalingam Priyanga , Hancock Kelly , Johnston Donna L. , Cataudella Danielle , Cassidy Marilyn , Punnett Angela S. , Shama Wendy
Published: December 2015

Background:

Best practice guidelines for the treatment of cancer now advocate for a child- and family-centered model of care and a psychosocial model of risk prevention. However, healthcare professionals (HCPs) report a number of barriers preventing the implementation of psychosocial care, including an absence of tools to help identify psychosocial problems within the family.

Objectives:

The aims of this study are to (1) explore the psychometric properties of the Psychosocial Care Checklist (PCCL) and (2) test if the PCCL can differentiate the degree to which HCPs are aware of psychosocial problems within the family (patient, siblings, parents) of a child with cancer.

Methods:

Thirty-seven HCPs caring for a child with cancer completed the PCCL at time 1 (2-4 weeks after diagnosis) and 29 HCPs completed the PCCL at time 2 (2-3 weeks after).

Results:

The PCCL had strong test-retest reliability for all domains (α > .60) and strong internal consistency for the total PCCL (α = .91). Interrater reliability was moderate for the oncologist-nurse dyad with regard to sibling knowledge (r = 0.56) and total psychosocial knowledge (r = 0.65). Social workers were significantly more knowledgeable than both nurses and oncologists about total family problems (P = .01) and sibling problems (P = .03).

Conclusions:

Preliminary findings suggest that the PCCL has adequate test-retest reliability and validity and is useful in differentiating the degree to which HCPs are aware of psychosocial problems within the family, with social workers being the most knowledgeable.

Implications for Practice:

Using the PCCL may help HCPs to identify psychosocial problems within the family and appropriately allocate psychosocial resources.
A systematic investigation of production of synthetic prions from recombinant prion protein
Authors: Schmidt Christian , Fizet Jeremie , Properzi Francesca , Batchelor Mark , Sandberg Malin K. , Edgeworth Julie A. , Afran Louise , Ho Sammy , Badhan Anjna , Klier Steffi , Linehan Jacqueline M. , Brandner Sebastian , Hosszu Laszlo L. P. , Tattum M. Howard , Jat Parmjit , Clarke Anthony R. , Klöhn Peter C. , Wadsworth Jonathan D. F. , Jackson Graham S. , Collinge John
Published: December 2015

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic
ex vivo
prions is solved.
Opposite sense ground rotations of a pair of Cavendish balances in earthquakes
Authors: Bourdillon Alain , Ropars Guy , Gaffet Stéphane , Le Floch Albert
Published: December 2015

Among the intermingled translational and rotational effects occurring in earthquakes, the translational effects are rather well understood. Recent experiments have been performed to investigate the rotational effects which have been observed for centuries that remain intriguing and less well understood. Although rotational seismology is of interest in a wide range of disciplines, rotational ground motions remain challenging to detect directly, especially their sense of rotation. To avoid a possible random response of a single balance, we locate two Cavendish balances in an ultra-low-noise laboratory. For the two successive 2012 Italian earthquakes in Emilia detected in exactly the same direction, opposed counterclockwise and clockwise responses of the two balances are recorded at the same site. Despite the complex combinations of Rayleigh and Love surface waves in the far-field, the two circular fundamental eigenmodes of the gravity-free torsion balances permit the determination of the ground rotation senses, which are indirectly confirmed by the phase shifted acceleration components in the balance frequency bandwidth, as well as the corresponding opposite driving angular impulses. The versatility of the Cavendish balances suggests that they could be used as inexpensive rotational sensor arrays in seismic areas to follow the propagation of ground rotations from the epicentres.
Dispersal as a source of variation in age-specific reproductive strategies in a wild population of lizards
Authors: Cotto Olivier , Massot Manuel , Ronce Ophélie , Clobert Jean
Published: December 2015

Dispersal syndromes describe the patterns of covariation of morphological, behavioural, and life-history traits associated with dispersal. Studying dispersal syndromes is critical to understanding the demographic and genetic consequences of movements. Among studies describing the association of life-history traits with dispersal, there is anecdotal evidence suggesting that dispersal syndromes can vary with age. Recent theory also suggests that dispersive and philopatric individuals might have different age-specific reproductive efforts. In a wild population of the common lizard (
Zootoca vivipara
), we investigated whether dispersive and philopatric individuals have different age-specific reproductive effort, survival, offspring body condition, and offspring sex ratio. Consistent with theoretical predictions, we found that young dispersive females have a higher reproductive effort than young philopatric females. Our results also suggest that the early high investment in reproduction of dispersive females trades-off with an earlier onset of senescence than in philopatric females. We further found that young dispersive females produce smaller offspring in lower body condition than do young philopatric females. Overall, our results provide empirical evidence that dispersive and philopatric individuals have different age-specific life-history traits.
Induced-fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids
Authors: Lehmann Jean
Published: December 2015

The catalytic site of most enzymes can essentially deal with only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the inducedfit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.