Interpenetrating network formation in agarose--kappa-carrageenan gel composites

Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the seaweed polysaccharides agarose and kappa-carrageenan. Over a range of ionic strength, and potassium content, there is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. At low ionic strength, where the agarose gels first, differential scanning calorimetry evidence shows some influence of the carrageenan on the agarose ordering enthalpy, particularly at higher polymer concentrations. As the potassium level is increased, however, and the order of gelling is reversed, this effect disappears. Cure behavior for the systems at high ionic strength can be described as a simple summation of the pure component contributions. At low ionic strength, on the other hand, the modulus behavior is more complex, suggesting either a modification, in the mixture, of the kappa-carrageenan gelling parameters or a more complex modulus additivity rule.     

BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.     

Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.