SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre‐implantation embryo development

ObjectivesGene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.Materials and methodsTo analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.ResultsOur results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.     

Electrochemical analysis of copper ion using a Gly–Gly–His tripeptide modified poly(3-thiopheneacetic acid) biosensor

A novel biosensor harnessing a conducting polymer functionalized with a copper ion specific peptide proved to be highly effective for electrochemical analysis of copper ions. The developed sensor comprised a transducer based on a conducting polymer (poly(3-thiopheneacetic acid)) electrode and a probe (tripeptide, Gly–Gly–His) selectively cognitive of copper ions. For functionalization of the electrode, the carboxylic group of the polymer was covalently coupled with the amine group of the tripeptide, and its structural features were confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection infrared (ATR-IR) spectroscopy. The peptide modified polythiophene biosensor was used for the electrochemical analysis of various trace metal ions by square wave voltammetry. The electrode was found to be highly sensitive and selective to Cu²⁺ in the range of 0.02–20 μM with almost no cross binding to other metal ions such as Ni²⁺ and Pb²⁺. Furthermore, the developed sensor exhibited a high stability and reproducibility despite the repeated use of the sensor electrode and probe. With the advent of more diverse affinity bioprobes specific towards a broad range of analytes, the demonstrated strategy harnessing peptide modified polythiophene biosensor is likely to provide an excellent platform for the selective determination of trace amount of analytes whose detection is otherwise cumbersome.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Background

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.

Methodology/Principal Findings

We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.

Conclusions/Significance

Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance.     

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.     

Non-parental infanticide in a dense population of the Black-billed Magpie (<Emphasis Type="Italic">Pica pica</Emphasis>)

We report the first case of non-parental infanticide in the Black-billed Magpie Pica pica. Using a video camera installed in the victims’ nest, we recorded repeated visits (over 4 days) of an adult (each time one bird) who attacked six nestlings at each visit until they died or were evicted. The nest was one of 58 nests filmed over four breeding seasons. Collected evidence suggests that the perpetrator(s) might have been the female breeder of the neighbouring nest, possibly also her male partner. The parental female aggressively attacked the perpetrator. Post-infanticide expansion of breeding territory by the suspected perpetrator is the hypothetical ultimate explanation of the observed infanticide. Movie clips from inside the nest are shown at: , , , and .     

Diet and ranging behavior of the endangered Javan gibbon (Hylobates moloch) in a submontane tropical rainforest

Altitude influences forest structure and food abundance and distribution, which in turn affect primate feeding and ranging patterns. Javan gibbons (Hylobates moloch) are endemic to forests spanning a broad range of altitudes on Java, Indonesia. Most information about Javan gibbon behavior comes from studies in lowland forests, while the vast majority of wild gibbons remaining inhabit hill and lower montane forests. We studied the diets, activity patterns, and ranging behavior of three gibbon groups in hill/lower montane (950-1,100?m asl) forest in the Gunung Halimun-Salak National Park (GHSNP) from April 2008 to March 2009. The mean home range size was 37?ha and the mean daily path length was 1,180?m. The study groups spent 36% of time feeding, 41% resting, 15% traveling, 6% engaging in social behavior, and 2% in aggressive interactions. Fruit was the most important food (63% of feeding time) followed by leaves (24%), and flowers (12%). Our results suggest that Javan gibbons in higher elevation habitats have substantially larger home ranges than lowland populations, despite broad similarity in their activity budgets and diets. Conservation managers should consider the effects of altitude and habitat quality on gibbon ranging behavior when developing habitat corridors, selecting sites for translocation or reintroduction projects, and designating and managing protected areas.