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Pennycress (Thlaspi arvense L.) is an annual cover crop known for its exceptional cold tolerance and high oil and protein yields. Pennycress can be integrated into a corn–soybean rotation in the U.S. However, the utilization of pennycress biomass remains largely unexplored, including assessing compositional changes through its growth and organic matter digestibility. This study harvested pennycress at three growth stages, characterized the biomass for anaerobic digestion (AD), and tested the effects of concurrent alkali pretreatment and ensiling on the biomass methane yield. Results showed that the biomass harvested when the plants were undergoing senescence (“third-harvest”) had higher contents of acid detergent fiber, neutral detergent fiber, and lignin, while the biomass harvested when 80–90% of the pods were fully-sized (“second-harvest”) had the highest protein content. The AD experiments showed that the first-harvest biomass (90% of flowers opened) failed to produce biogas due to a drop in the pH and alkalinity, the second-harvest biomass was inhibited for methane production (45.74 ± 0.20 L/kg-VS), and the third-harvest biomass had a methane yield of 171.80 ± 4.82 L/kg-VS. After the alkali pretreatment and ensiling, a methane yield of 270.4 ± 3.10 L/kg-VS was obtained from the second-harvest biomass, representing a significant 4.5-fold increase (adjusted for the organic matter loss) relative to the untreated second-harvest biomass.

Summary: Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle. [ABSTRACT FROM AUTHOR]

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher ( P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 104 copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model. [ABSTRACT FROM AUTHOR]

Abstract: The myosin heavy chain gene, MYH M743 - 2 , is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5''-flanking region of MYH M743 - 2 into fertilized eggs of zebrafish and medaka. Although the -2.1kb 5''-flanking region of torafugu MYH M743 - 2 showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5''-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1kb 5''-flanking region of MYH M743 - 2 contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka. [Copyright &y& Elsevier]

Intracellular targeting of the Pseudomonas aeruginosa toxins, such as exoenzyme S (ExoS), cause cell death, as well as morphological and physiological changes in various tissue culture cells and animal models. In this report we have investigated the mechanism behind ExoS-mediated cell death. In order to address this issue, we have used cell lines expressing activated forms of various components of the Ras signalling pathway in order to evaluate the importance of the Ras pathway for viability and survival upon ExoS infection. Here we show that activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. Further, an activated form of protein kinase B (PKB)/Akt also leads to decreased level of cell death in response to ExoS infection, indicating that an important ExoS survival target is located upstream of Raf-1 and PKB/Akt. Moreover, we show that ExoS infection inhibits phosphorylation of FOXO3a, and induces caspase-3 activity, which are hallmarks for induction of cell death. In conclusion, we suggest that Ras proteins are an important cellular target for the P. aeruginosa toxin ExoS, which induces cell death during pathogenesis as a means of defending the bacterium against eukaryotic phagocytosis. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the effectiveness of ciprofloxacin treatment for Neisseria gonorrhoeae among female sex workers in Dhaka, Bangladesh. Methodology; Results of the study; Discussion.

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use., Version of Record

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175-131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.