bluuebunny/crossref_metadata_2025 huggingface dataset:
61 DIPLOID (BOS TAURUS) AND TETRAPLOID (BOS INDICUS) EMBRYO AGGREGATION TO PRODUCE BOVINE EMBRYONIC CHIMERAS
Authors: Montanari Razza E. , Satrapa R. A. , Emanuelli I. P. , Moraes Barros C. , Nogueira M. F. G.
Published: December 2011

The formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components) would be mostly from the zebu embryo, which could interact with the taurine embryo/fetus in a different way (than it occurs in whole Bos taurus conceptus) during pregnancy in a tropical environment. The purpose of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production of embryonic chimeras by aggregation of 1/2 B. taurus (2n) with B. indicus (4n) embryos. Oocytes from Nelore cows from the abattoir were matured, fertilized with semen from Nelore and Holstein bulls and cultured in SOF. Two-cell stage Nelore embryos (30 h post-insemination), with a well-defined inter-blastomeric axis, were selected for the electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce 4n embryos. For this procedure, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140 and 500 V) and duration of electroshock (20, 25, 50 and 60 μs). Nelore 4n embryos produced after electrofusion and 2n taurine embryos, both at 8 to 16-cell stages (72 h post-insemination) were subjected to protease treatment to remove the zona pellucida and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and B. taurus (2n) embryos were added in pairs (4n+2n) into individual wells (well of the well, WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested parameters, the best fusion results (92%) and rates of cleavage after fusion (66%) were obtained with a single pulse of 75 V for 60 μs. The production rate of expanded 4n blastocysts was 31.5% using these parameters. After 4 replicates (still in the final stage of standardization), 4 blastocyst chimeras (4n+2n) were obtained from 31 attempts (13%). The production of bovine embryonic chimeras [Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique in applied research, by producing exclusively taurine calves, but with placental elements from the B. indicus breed, following transfer of these chimeras into recipient cows.
Financial support received from FAPESP, Brazil.
72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION
Authors: de Souza D. M. , Fernandes H. , Silva P. V. , Cazari B. , Moço P. D. , Campanha B. C. S. , Emanuelli I. P. , Nogueira M. F. G.
Published: December 2011

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components.
Financial support was received from FAPESP of Brazil.
77 REPRODUCTIVE PERFORMANCE OF DAIRY COWS DIFFERING IN THE INTERVAL FROM CALVING TO THE FIRST OVULATION IN THE POSTPARTUM PERIOD
Authors: Yamaguchi M. , Takahashi S. , Koyama H. , Dochi O.
Published: December 2011

In recent years, reproductive performance of dairy cows has declined worldwide. The reproductive characteristics of dairy cows should be evaluated to improve their fertility. Dochi et al. (2010) had reported that dairy cows that exhibited early first ovulation gave a low milk yield, had an early recovery of body condition score (BCS) in the postpartum period and a shorter number of days open. The objective of this study was to evaluate the effect of BCS on the functional recovery of reproduction after the birth of the calves. In this study, lactating Holstein cows were placed in free-stall housing equipped with an automatic milking system. The data presented in this study were collected from 48 cows that had calved between December 2007 and October 2010 with an average parity of 3.4 (range 2–6). The reproductive performance and the BCS during the different intervals between calving and the first ovulation were analysed using ANOVA. The conception rate at the first service was 38.1%. The average milk yield (305 days) was 12 299 kg. The first ovulation and oestrus were determined by ultrasonography. The results of the different intervals to the first ovulation are presented in Table 1. The average interval from the start of days in milk (DIM) to the first ovulation, first oestrus and first service; the number of days open; the number of services per conception and the milk yield (kg day–1, by 90 DIM) were 37.4, 54.7 and 110.3 days; 168.2 days open; 2.5 times; and 42.1 kg day–1, respectively. The cows that exhibited early first ovulation (≤21 DIM) in the postpartum period had significantly shorter intervals between the start of DIM and the first oestrus (P < 0.01). However, the days to first service, the number of days open, the number of services per conception and the milk yield (kg day–1, by 90 DIM) did not differ significantly. The BCS in the peripartum period of different intervals to the first ovulation decreased from 3.1 to 2.4 by 150 DIM. This study showed that average BCS of preparturient 50–10 DIM is 3.0. Moreover, the average BCS of parturition is declining to 2.5 (parturition from 70 to 150 DIM). The cows became pregnant after an increase in the BCS (>150 DIM). In conclusion, these results suggest that the delay to the first ovulation in the postpartum period tended to have delayed the oestrus expression in the cows. Moreover, the low level of BCS in the peripartum period, regardless of the number of days of the first ovulation and the first oestrus, potentially influenced the prolongation in the number of days open. To improve the fertility of the cows, it is important to elucidate the factors affecting the fluctuations in the BCS during the peripartum period.

Table 1.Reproductive performance of dairy cows differing in interval to postpartum first ovulation
8 EFFECT OF SUPPLEMENTATION WITH ESTRADIOL OR ESTRADIOL PLUS GONADOTROPIN-RELEASING HORMONE AFTER PROSTAGLANDIN F2α ADMINISTRATION ON THE PREGNANCY RATES FOLLOWING TIMED ARTIFICIAL INSEMINATION IN DAIRY COWS
Authors: Jeong J. K. , Kang H. G. , Kim I. H.
Published: December 2011

This study determined the effect of supplementation with oestradiol or oestradiol plus GnRH after PGF2α administration on pregnancy rates after timed artificial insemination (TAI) in dairy cows. Three hundred and six Holstein dairy cows with corpus luteum confirmed by ultrasonography (Tringa Linear with 5.0 MHz array transducer; Esaote Pie Medical, Maastricht, the Netherlands) were randomly allocated to three treatments: a single injection of 500 μg of cloprostenol, PGF2α analogue (Estrumate, Schering-Plough Animal Health, Friesoythe, Germany; PGF2α group, n = 115) designated as 0 h, an injection of 500 μg of cloprostenol and an injection of 1 mg of oestradiol benzoate (EB, SY Esrone; Samyang, Seoul, Korea) 56 h later (PGF2α + EB group, n = 114), or the same treatment as in the PGF2α + EB group and an additional injection of 100 μg of gonadorelin, GnRH analogue (Godorel, Uni-Biotech Co., Ltd., Korea) 24 h later (PGF2α + EB + GnRH group, n = 77). All cows in each group received TAI 80 h after the PGF2α injection. Pregnancy was determined at 40 to 50 days after TAI by using both ultrasonography and rectal palpation. Pregnancy rate among groups were compared by the chi-square test using the SAS program (version 9.1: SAS Institute Inc., Cary, NC, USA). Pregnancy rate following TAI were higher (P < 0.05) in the PGF2α + EB + GnRH group (34/77, 44.2%) than in the PGF2α group (31/115, 27.0%), with the PGF2α + EB group intermediate (39/114, 34.2%). In conclusion, the supplementation with oestradiol plus GnRH after PGF2α administration improved pregnancy rate after TAI in dairy cows.
This work was supported by the research grant of the Chungbuk National University in 2011.
82 EXPRESSION OF CELL MEMBRANE Mg2+ INORGANIC PHOSPHATE CHANNELS IN THE HUMAN PLACENTA DERIVED FROM PREGNANT WOMEN AND HUMAN PLACENTAL CELL LINES (BeWo, JEG3, hPC)
Authors: Yang H. , Hyun S. H. , Jeung E. B.
Published: December 2011

Preeclampsia is a pregnancy-specific disease characterised by de novo development of concurrent hypertension and oxidative stress in the placenta. The human placenta is a highly vascularized organ whose major function is to allow maternal–fetal exchange of solutes such as Ca2+ and oxygen. The transient receptor potential cation channel subfamily is known to contain channels activated by such various stimuli as intracellular Ca2+ membrane potential, cold and pH. However, signalling mechanisms mediating hormonal regulation of Mg2+ inorganic phosphate channels during the placenta duration of pregnancy are incompletely understood. We examined the expression of cell membrane Mg2+ inorganic channels in 3 sections (fetal-, central-, maternal-) of preeclamptic placenta (PEP) and from placental cell lines, BeWo, JEG3 and hPC (isolated during the first trimester) by real-time PCR and Western blot analysis. Placental tissues (∼3 cm) from women (n = 75) between the ages of 28 to 45 years undergoing normal or Caesarean delivery were dissected into 1-cm sections. Data were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. P < 0.05 was considered to be statistically significant. During preterm labour, human placental expression of Mg2+ inorganic channel mRNA and protein fluctuated in the 3 sections of PEP compared with normal placenta. In hPC, the expression of Mg2+ inorganic channel (TRPM6/7, MagT1, NIPA2, SLC41A1 to 3 and SLC34A1 to 3) mRNA and protein were decreased in placental cell lines by hypoxic stress (2% O2/93% N2/5% CO2) compared with normoxia (20% O2/75% N2/5% CO2). The levels of Mg2+ inorganic channel mRNA and protein were distinctly expressed between BeWo and JEG3 cells. These results indicate that placental Mg2+ inorganic channels play potential roles in differential sections of placenta between normal and PEP, suggesting that induced Mg2+ inorganic channels of PEP may be involved in preeclamptic stress in human placenta and placental cell lines, which are a determinant factor affecting calcium transfer.
91 INTERACTION OF OXYGEN TENSION AND CYSTEAMINE SUPPLEMENTATION DURING IN VITRO CULTURE OF BOVINE EMBRYOS
Authors: Stauber L. M. , Seidel G. E.
Published: December 2011

Reactive oxygen species damage early mammalian embryos, so culture of bovine in vitro-produced (IVP) embryos at 5% O2 is clearly superior to 20% O2. The thiol compound cysteamine is an antioxidant and improves in vitro blastocyst production when added to in vitro maturation (IVM) medium. The purpose of this study was to investigate supplementation of IVP embryo culture medium with cysteamine at different oxygen tensions. Bovine ovaries from feedlot heifers were collected from a local abattoir and cumulus–oocyte complexes (COC) were aspirated from 3- to 8-mm follicles. COC were matured in maturation medium with 100μM cysteamine (Reprod. Fertil. Dev. 18, 585) for 23 h in a humidified incubator at 38.5°C in 5% CO2 in air. COC at 23 h of maturation were co-incubated with sperm for 18 h. Cumulus cells were then removed and presumed zygotes were cultured in our chemically defined culture medium system (Reprod. Fertil. Dev. 18, 585) in a 2 × 2 factorial arrangement: with or without 50 μM cysteamine × atmospheres of either 5% O2, 5% CO2, 90% N2, or 5% CO2 in air. There were no treatment differences (P > 0.01) in percentages of oocytes cleaving or reaching the 8-cell stage (Table 1). However, blastocyst production rates were lower (P < 0.01) in the group cultured without cysteamine at 20% O2 compared with all the other groups. Adding cysteamine for embryo culture at 20% O2 resulted in blastocyst rates similar to those cultured at 5% O2 with or without cysteamine. Cysteamine was not beneficial at 5% O2.

Table 1.Cysteamine supplementation during in vitro culture of bovine embryos
98 EFFECT OF ZONA PELLUCIDA ON PORCINE PARTHENOGENETICALLY ACTIVATED EMBRYOS
Authors: Li R. , Liu Y. , Li J. , Kragh P. M. , Callesen H.
Published: December 2011

The need for zona pellucida (ZP) during pre-implantation embryo development is still debated. In porcine parthenogenetically activated (PA) embryos, we have previously shown a different distribution in cell numbers on Day 6 blastocysts cultured with or without ZP (Li et al. 2010 Reprod. Fertil. Dev. 22, 234). In the present study, we expanded this study to include also the timing of early development and the resulting quality and robustness (for vitrification) of porcine PA embryos. Parthenogenetic activation was made first by an electric pulse (1.26 kV cm–1, 80 μs) and then by incubation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium for 4 h. ZP was removed by 3.3 mg mL–1 pronase. Both zona-intact (PAZI) and zona-free (PAZF) embryos were cultured individually for 6 days either in time-lapse incubator (Embryoscope D, Unisense A/S, Aarhus, Denmark) for 15-min observations (Exp. 1; 60 oocytes, 2 replicates) or in standard a incubator for blastocyst quality studies on Day 6 (Exp. 2; 524 oocytes, 11 replicates) or for cryo-tolerance studies with vitrification using Cryotop on Day 4, followed by warming and 2 days further culture (Exp. 3; 449 oocytes, 4 replicates). The timing of morulae was recorded when they completed compaction. Good blastocysts were defined when they expanded to 1.5 times larger than oocytes and formed regular blastocoel cavity with uniform colour and distribution of cells. Timing data were analysed by Student's t-test, while development rates and survival rates were analysed by chi-squared test. Exp. 1: after activation, 42 blastocysts formed on Day 6, during which the timing of development was monitored (Table 1). PAZF embryos developed faster than PAZI, especially during the first 3 cell cycles. Exp. 2: after activation, 212 and 197 blastocysts formed on Day 6 with or without ZP, respectively. Both rates of total blastocysts and good blastocysts of PAZI embryos were significantly higher than those of PAZF embryos (80.1 ± 2.7% vs 69.9 ± 1.1%, 61.9 ± 3.3% vs 49.5 ± 2.5%, respectively), but no difference was found in all blastocyst's cell numbers between PAZI and PAZF (48.2 ± 2.3 and 47.9 ± 3.2, respectively). Exp. 3: after activation, 107 PAZI and 44 PAZF embryos were vitrified on Day 4. More PAZI than PAZF embryos survived (60.8 ± 8.3% vs 30.4 ± 11.9%; P < 0.05). In conclusion, removal of ZP can increase the speed of development of porcine PA embryos, especially at the timing of embryonic genome activation (5-cell stage). Furthermore, the zona pellucida can benefit the blastocyst formation and cryo-tolerance for PA embryos, perhaps by creating a more stable microenvironement.

Table 1.Timing of developmental stages of porcine PA embryos with (PAZI) or without (PAZF) zona pellucida*
99 DIFFERENTIAL GENE REGULATION OF STEROIDOGENIC TRANSCRIPTS AND ESTRADIOL PRODUCTION FOLLOWING IN VITRO PIG EMBRYO ELONGATION IN ALGINATE HYDROGEL THREE-DIMENSIONAL MATRIX
Authors: Miles J. R. , Sargus C. N. , Plautz S. A. , Vallet J. L. , Pannier A. K.
Published: December 2011

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production.
†USDA is an equal opportunity provider and employer.
Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides
Authors: Neef Anne B. , Luedtke Nathan W.
Published: December 2011

Commonly used metabolic labels for DNA, including 5-ethynyl-2′-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosyl-ethynyluracil derivatives. Among these, (2′
S
)-2′-deoxy-2′-fluoro-5-ethynyluridine (F-
ara
-EdU) exhibited selective DNA labeling, yet had a minimal impact on genome function in diverse tissue types. Metabolic incorporation of F-
ara
-EdU into DNA was readily detectable using copper(I)-catalyzed azide–alkyne “click” reactions with fluorescent azides. F-
ara
-EdU is less toxic than both BrdU and EdU, and it can be detected with greater sensitivity in experiments where long-term cell survival and/or deep-tissue imaging are desired. In contrast to previously reported 2′-arabino modified nucleosides and EdU, F-
ara
-EdU causes little or no cellular arrest or DNA synthesis inhibition. F-
ara
-EdU is therefore ideally suited for pulse-chase experiments aimed at “birth dating” DNA in vivo. As a demonstration, Zebrafish embryos were microinjected with F-
ara
-EdU at the one-cell stage and chased by BrdU at 10 h after fertilization. Following 3 d of development, complex patterns of quiescent/senescent cells containing only F-
ara
-EdU were observed in larvae along the dorsal side of the notochord and epithelia. Arabinosyl nucleoside derivatives therefore provide unique and effective means to introduce bioorthogonal functional groups into DNA for diverse applications in basic research, biotechnology, and drug discovery.
Ouabain modulates ciliogenesis in epithelial cells
Authors: Larre Isabel , Castillo Aida , Flores-Maldonado Catalina , Contreras Ruben G. , Galvan Ivan , Muñoz-Estrada Jesus , Cereijido Marcelino
Published: December 2011

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na
+
and H
2
O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na
+
,K
+
-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.
Structural insights into protein arginine symmetric dimethylation by PRMT5
Authors: Sun Litao , Wang Mingzhu , Lv Zongyang , Yang Na , Liu Yingfang , Bao Shilai , Gong Weimin , Xu Rui-Ming
Published: December 2011

Symmetric and asymmetric dimethylation of arginine are isomeric protein posttranslational modifications with distinct biological effects, evidenced by the methylation of arginine 3 of histone H4 (H4R3): symmetric dimethylation of H4R3 leads to repression of gene expression, while asymmetric dimethylation of H4R3 is associated with gene activation. The enzymes catalyzing these modifications share identifiable sequence similarities, but the relationship between their catalytic mechanisms is unknown. Here we analyzed the structure of a prototypic symmetric arginine dimethylase, PRMT5, and discovered that a conserved phenylalanine in the active site is critical for specifying symmetric addition of methyl groups. Changing it to a methionine significantly elevates the overall methylase activity, but also converts PRMT5 to an enzyme that catalyzes both symmetric and asymmetric dimethylation of arginine. Our results demonstrate a common catalytic mechanism intrinsic to both symmetric and asymmetric arginine dimethylases, and show that steric constrains in the active sites play an essential role in determining the product specificity of arginine methylases. This discovery also implies a potentially regulatable outcome of arginine dimethylation that may provide versatile control of eukaryotic gene expression.
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant
Authors: Zeitlin Larry , Pettitt James , Scully Corinne , Bohorova Natasha , Kim Do , Pauly Michael , Hiatt Andrew , Ngo Long , Steinkellner Herta , Whaley Kevin J. , Olinger Gene G.
Published: December 2011

No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED50= 33 μg). A version with typical heterogenous mammalian glycoforms (ED50= 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complexN-glycosylation lacking core fucose exhibited superior potency (ED50= 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of FcN-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.
Increased expression of histone demethylase JHDM1D under nutrient starvation suppresses tumor growth via down-regulating angiogenesis
Authors: Osawa Tsuyoshi , Muramatsu Masashi , Wang Feng , Tsuchida Rika , Kodama Tatsuhiko , Minami Takashi , Shibuya Masabumi
Published: December 2011

Histone demethylase JHDM1D (also known as KDM7A) modifies the level of methylation in histone and participates in epigenetic gene regulation; however, the role of JHDM1D in tumor progression is unknown. Here, we show that JHDM1D plays a tumor-suppressive role by regulating angiogenesis. Expression of JHDM1D was increased in mouse and human cancer cells under long-term nutrient starvation in vitro. Expression of
JHDM1D
mRNA was increased within avascular tumor tissue at the preangiogenic switch, along with increased expression of angiogenesis-regulating genes such as
Vegf-A
. Stable expression of JHDM1D cDNA or siRNA silencing of JHDM1D in cancer cells did not affect cell proliferation, anchorage-independent cell growth, or cell cycle progression in vitro. Notably, JHDM1D-expressing mouse melanoma (B16) and human cervical carcinoma (HeLa) cells exhibited significantly slower tumor growth in vivo compared with the original cells. This reduction in tumor growth was associated with decreased formation of CD31
+
blood vessels and reduced infiltration of CD11b
+
macrophage linage cells into tumor tissues. Expression of multiple angiogenic factors such as
VEGF-B
and angiopoietins was decreased in tumor xenografts of JHDM1D-expressing B16 and HeLa cells. Our results provide evidence that increased JHDM1D expression suppressed tumor growth by down-regulating angiogenesis under nutrient starvation.
Regulation of phenotypic heterogeneity permits
Salmonella
evasion of the host caspase-1 inflammatory response
Authors: Stewart Mary K. , Cummings Lisa A. , Johnson Matthew L. , Berezow Alex B. , Cookson Brad T.
Published: December 2011

Sensing and adapting to the environment is one strategy by which bacteria attempt to maximize fitness in an unpredictable world; another is the stochastic generation of phenotypically distinct subgroups within a genetically clonal population. In culture,
Salmonella
Typhimurium populations are bistable for the expression of flagellin. We report that YdiV controls this expression pattern by preventing transcription of the sigma factor that recruits RNA polymerase to the flagellin promoter. Bistability ensues when the sigma factor is repressed in a subpopulation of cells, resulting in two phenotypes: flagellin expressors and flagellin nonexpressors. Although the ability to swim is presumably a critical survival trait, flagellin activates eukaryotic defense pathways, and
Salmonella
restrict the production of flagellin during systemic infection.
Salmonella
mutants lacking YdiV are unable to fully repress flagellin at systemic sites, rendering them vulnerable to caspase-1 mediated colonization restriction. Thus, a regulatory mechanism producing bistability also impacts
Salmonella
virulence.
Rice consumption contributes to arsenic exposure in US women
Authors: Gilbert-Diamond Diane , Cottingham Kathryn L. , Gruber Joann F. , Punshon Tracy , Sayarath Vicki , Gandolfi A. Jay , Baker Emily R. , Jackson Brian P. , Folt Carol L. , Karagas Margaret R.
Published: December 2011

Emerging data indicate that rice consumption may lead to potentially harmful arsenic exposure. However, few human data are available, and virtually none exist for vulnerable periods such as pregnancy. Here we document a positive association between rice consumption and urinary arsenic excretion, a biomarker of recent arsenic exposure, in 229 pregnant women. At a 6-mo prenatal visit, we collected a urine sample and 3-d dietary record for water, fish/seafood, and rice. We also tested women's home tap water for arsenic, which we combined with tap water consumption to estimate arsenic exposure through water. Women who reported rice intake (
n
= 73) consumed a median of 28.3 g/d, which is ∼0.5 cup of cooked rice each day. In general linear models adjusted for age and urinary dilution, both rice consumption (g, dry mass/d) and arsenic exposure through water (μg/d) were significantly associated with natural log-transformed total urinary arsenic (

,

, both
P
< 0.0001), as well as inorganic arsenic, monomethylarsonic acid, and dimethylarsinic acid (each
P
< 0.005). Based on total arsenic, consumption of 0.56 cup/d of cooked rice was comparable to drinking 1 L/d of 10 μg As/L water, the current US maximum contaminant limit. US rice consumption varies, averaging ∼0.5 cup/d, with Asian Americans consuming an average of >2 cups/d. Rice arsenic content and speciation also vary, with some strains predominated by dimethylarsinic acid, particularly those grown in the United States. Our findings along with others indicate that rice consumption should be considered when designing arsenic reduction strategies in the United States.
Phytophthora infestanseffector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface
Authors: Bozkurt Tolga O. , Schornack Sebastian , Win Joe , Shindo Takayuki , Ilyas Muhammad , Oliva Ricardo , Cano Liliana M. , Jones Alexandra M. E. , Huitema Edgar , van der Hoorn Renier A. L. , Kamoun Sophien
Published: December 2011

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogenPhytophthora infestansfocally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants toP. infestansby targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility toP. infestansin a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.
Local adaptation of an introduced transgenic insect fungal pathogen due to new beneficial mutations
Authors: Wang Sibao , O’Brien Tammatha R. , Pava-Ripoll Monica , St. Leger Raymond J.
Published: December 2011

Genetically modified
Metarhizium
spp represent a major new arsenal for combating insect pests and insect-borne diseases. However, for these tools to be used safely and effectively, we need a much better understanding of their evolutionary potential and invasion ecology. In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of
Metarhizium robertsii
following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the new mutations increased the fitness of
M. robertsii
in the new range by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. Our results provide a mechanistic basis for understanding postrelease adaptations, show that agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the
Mad2
gene.
Adding a dimension to the infrared spectra of interfaces using heterodyne detected 2D sum-frequency generation (HD 2D SFG) spectroscopy
Authors: Xiong Wei , Laaser Jennifer E. , Mehlenbacher Randy D. , Zanni Martin T.
Published: December 2011

In the last ten years, two-dimensional infrared spectroscopy has become an important technique for studying molecular structures and dynamics. We report the implementation of heterodyne detected two-dimensional sum-frequency generation (HD 2D SFG) spectroscopy, which is the analog of 2D infrared (2D IR) spectroscopy, but is selective to noncentrosymmetric systems such as interfaces. We implement the technique using mid-IR pulse shaping, which enables rapid scanning, phase cycling, and automatic phasing. Absorptive spectra are obtained, that have the highest frequency resolution possible, from which we extract the rephasing and nonrephasing signals that are sometimes preferred. Using this technique, we measure the vibrational mode of CO adsorbed on a polycrystalline Pt surface. The 2D spectrum reveals a significant inhomogenous contribution to the spectral line shape, which is quantified by simulations. This observation indicates that the surface conformation and environment of CO molecules is more complicated than the simple “atop” configuration assumed in previous work. Our method can be straightforwardly incorporated into many existing SFG spectrometers. The technique enables one to quantify inhomogeneity, vibrational couplings, spectral diffusion, chemical exchange, and many other properties analogous to 2D IR spectroscopy, but specifically for interfaces.
Energy landscape of the reactions governing the Na
+
deeply occluded state of the Na
+
/K
+
-ATPase in the giant axon of the Humboldt squid
Authors: Castillo Juan P. , De Giorgis Daniela , Basilio Daniel , Gadsby David C. , Rosenthal Joshua J. C. , Latorre Ramon , Holmgren Miguel , Bezanilla Francisco
Published: December 2011

The Na
+
/K
+
pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na
+
from the cell and import 2K
+
per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na
+
/K
+
pump that represent the kinetics of extracellular Na
+
binding/release and Na
+
occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid
Dosidicus gigas
, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na
+
from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.
Electronic dynamics and plasmons of sodium under compression
Authors: Mao Ho-Kwang , Ding Yang , Xiao Yuming , Chow Paul , Shu Jinfu , Lebègue Sébastien , Lazicki Amy , Ahuja Rajeev
Published: December 2011

Sodium, which has long been regarded as one of the simplest metals, displays a great deal of structural, optical, and electronic complexities under compression. We compressed pure Na in the body-centered cubic structure to 52 GPa and in the face-centered cubic structure from 64 to 97 GPa, and studied the plasmon excitations of both structures using the momentum-dependent inelastic X-ray scattering technique. The plasmon dispersion curves as a function of pressure were extrapolated to zero momentum with a quadratic approximation. As predicted by the simple free-electron model, the square of the zero-momentum plasmon energy increases linearly with densification of the body-centered cubic Na up to 1.5-fold. At further compressions and in face-centered cubic Na above 64 GPa, the linear relation curves progressively toward the density axis up to 3.7-fold densification at 97 GPa. Ab initio calculations indicate that the deviation is an expected behavior of Na remaining a simple metal.
Nonlinear detection of paleoclimate-variability transitions possibly related to human evolution
Authors: Donges Jonathan F. , Donner Reik V. , Trauth Martin H. , Marwan Norbert , Schellnhuber Hans-Joachim , Kurths Jürgen
Published: December 2011

Potential paleoclimatic driving mechanisms acting on human evolution present an open problem of cross-disciplinary scientific interest. The analysis of paleoclimate archives encoding the environmental variability in East Africa during the past 5 Ma has triggered an ongoing debate about possible candidate processes and evolutionary mechanisms. In this work, we apply a nonlinear statistical technique, recurrence network analysis, to three distinct marine records of terrigenous dust flux. Our method enables us to identify three epochs with transitions between qualitatively different types of environmental variability in North and East Africa during the (i) Middle Pliocene (3.35–3.15 Ma B.P.), (ii) Early Pleistocene (2.25–1.6 Ma B.P.), and (iii) Middle Pleistocene (1.1–0.7 Ma B.P.). A deeper examination of these transition periods reveals potential climatic drivers, including (i) large-scale changes in ocean currents due to a spatial shift of the Indonesian throughflow in combination with an intensification of Northern Hemisphere glaciation, (ii) a global reorganization of the atmospheric Walker circulation induced in the tropical Pacific and Indian Ocean, and (iii) shifts in the dominating temporal variability pattern of glacial activity during the Middle Pleistocene, respectively. A reexamination of the available fossil record demonstrates statistically significant coincidences between the detected transition periods and major steps in hominin evolution. This result suggests that the observed shifts between more regular and more erratic environmental variability may have acted as a trigger for rapid change in the development of humankind in Africa.
Mouse digit tip regeneration is mediated by fate-restricted progenitor cells
Authors: Lehoczky Jessica A. , Robert Benoît , Tabin Clifford J.
Published: December 2011

Regeneration of appendages is frequent among invertebrates as well as some vertebrates. However, in mammals this has been largely relegated to digit tip regeneration, as found in mice and humans. The regenerated structures are formed from a mound of undifferentiated cells called a blastema, found just below the site of amputation. The blastema ultimately gives rise to all of the tissues in the regenerate, excluding the epidermis, and has classically been thought of as a homogenous pool of pluripotent stem cells derived by dedifferentiation of stump tissue, although this has never been directly tested in the context of mammalian digit tip regeneration. Successful digit tip regeneration requires that the level of amputation be within the nail bed and depends on expression of Msx1. Because Msx1 is strongly expressed in the nail bed mesenchyme, it has been proposed that the Msx1-expressing cells represent a pluripotent cell population for the regenerating digit. In this report, we show that Msx1 is dynamically expressed during digit tip regeneration, and it does not mark a pluripotent stem cell population. Moreover, we show that both the ectoderm and mesoderm contain fate-restricted progenitor populations that work in concert to regenerate their own lineages within the digit tip, supporting the hypothesis that the blastema is a heterogeneous pool of progenitor cells.
Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors
Authors: Janes Peter W. , Griesshaber Bettina , Atapattu Lakmali , Nievergall Eva , Hii Linda L. , Mensinga Anneloes , Chheang Chanly , Day Bryan W. , Boyd Andrew W. , Bastiaens Philippe I. , Jørgensen Claus , Pawson Tony , Lackmann Martin
Published: December 2011

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph–Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.
Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites
Authors: Gong Jingyi , Sun Zhiqi , Wu Lizhen , Xu Wenyi , Schieber Nicole , Xu Dijin , Shui Guanghou , Yang Hongyuan , Parton Robert G. , Li Peng
Published: December 2011

Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD–LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.
Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome
Authors: Ouchi Takeshi , Kubo Akiharu , Yokouchi Mariko , Adachi Takeya , Kobayashi Tetsuro , Kitashima Daniela Y. , Fujii Hideki , Clausen Björn E. , Koyasu Shigeo , Amagai Masayuki , Nagao Keisuke
Published: December 2011

Epidermal Langerhans cells (LCs) extend dendrites through tight junctions (TJs) to survey the skin surface, but their immunological contribution in vivo remains elusive. We show that LCs were essential for inducing IgG1 responses to patch-immunized ovalbumin in mice that lacked skin dendritic cell subsets. The significance of LC-induced humoral responses was demonstrated in a mouse model of staphylococcal scalded skin syndrome (SSSS), a severe blistering disease in which the desmosomal protein Dsg1 (desmoglein1) is cleaved by Staphylococcus aureus–derived exfoliative toxin (ET). Importantly, ET did not penetrate TJs, and patch immunization did not alter epidermal integrity. Nevertheless, neutralizing anti-ET IgG1 was induced after patch immunization and abolished upon LC depletion, indicating that antigen capture through TJs by LCs induced humoral immunity. Strikingly, the ET-patched mice were protected from developing SSSS after intraperitoneal ET challenge, whereas LC-depleted mice were susceptible to SSSS, demonstrating a vital role for LC-induced IgG1 in systemic defense against circulating toxin in vivo. Therefore, LCs elicit humoral immunity to antigens that have not yet violated the epidermal barrier, providing preemptive immunity against potentially pathogenic skin microbes. Targeting this immunological process confers protection with minimal invasiveness and should have a marked impact on future strategies for development of percutaneous vaccines.