Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis

Xylose reductase (XR) is the first enzyme in D: -xylose metabolism, catalyzing the reduction of D: -xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)(-1)), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L(-1) h(-1) and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L(-1) h(-1); yield 59%).     

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle^−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.     

Evaluation of the radiation response and regenerative effects of mesenchymal stem cell-conditioned medium in an intestinal organoid system

Intestinal organoids have recently emerged as an in vitro model relevant to the gut system owing to their recapitulation of the native intestinal epithelium with crypt–villus architecture. However, it is unclear whether intestinal organoids reflect the physiology of the in vivo stress response. Here, we systemically investigated the radiation response in organoids and animal models using mesenchymal stem cell-conditioned medium (MSC-CM), which contains secreted paracrine factors. Irradiated organoids exhibited sequential induction of viability loss and regrowth after irradiation (within 12 days), similar to the response of the native intestinal epithelium. Notably, treatment with MSC-CM facilitated the reproliferation of intestinal stem cells (ISCs) and restoration of damaged crypt-villus structures in both models. Furthermore, Wnt/Notch signaling pathways were commonly upregulated by MSC-CM, but not radiation, and pharmacologically selective inhibition of Wnt or Notch signaling attenuated the enhanced recovery of irradiated organoids, with increases in ISCs, following MSC-CM treatment. Interestingly, the expression of Wnt4, Wnt7a, and active β-catenin was increased, but not notch family members, in MSC-CM-treated organoid after irradiation. Treatment of recombinant mouse Wnt4 and Wnt7a after irradiation improved to some extent intestinal epithelial regeneration both in vitro and in vivo. Overall, these results suggested that intestinal organoids recapitulated the physiological stress response of the intestinal epithelium in vivo. Thus, our findings provided important insights into the physiology of intestinal organoids and may contribute to the development of strategies to enhance the functional maturation of engineered organoids.     

Quantitative Tumor Segmentation for Evaluation of Extent of Glioblastoma Resection to Facilitate Multisite Clinical Trials

Standard-of-care therapy for glioblastomas, the most common and aggressive primary adult brain neoplasm, is maximal safe resection, followed by radiation and chemotherapy. Because maximizing resection may be beneficial for these patients, improving tumor extent of resection (EOR) with methods such as intraoperative 5-aminolevulinic acid fluorescence-guided surgery (FGS) is currently under evaluation. However, it is difficult to reproducibly judge EOR in these studies due to the lack of reliable tumor segmentation methods, especially for postoperative magnetic resonance imaging (MRI) scans. Therefore, a reliable, easily distributable segmentation method is needed to permit valid comparison, especially across multiple sites. We report a segmentation method that combines versatile region-of-interest blob generation with automated clustering methods. We applied this to glioblastoma cases undergoing FGS and matched controls to illustrate the method's reliability and accuracy. Agreement and interrater variability between segmentations were assessed using the concordance correlation coefficient, and spatial accuracy was determined using the Dice similarity index and mean Euclidean distance. Fuzzy C-means clustering with three classes was the best performing method, generating volumes with high agreement with manual contouring and high interrater agreement preoperatively and postoperatively. The proposed segmentation method allows tumor volume measurements of contrast-enhanced T₁-weighted images in the unbiased, reproducible fashion necessary for quantifying EOR in multicenter trials.     

Long-term prognosis of symptomatic isolated middle cerebral artery disease in Korean stroke patients

Background  

This study aimed to investigate the long-term mortality and recurrence rate of stroke in first-time stroke patients with symptomatic isolated middle cerebral artery disease (MCAD) under medical management.     

Variations in ozone-sensitivity in relation to cell cycle and ploidy in Saccharomyces cerevisiae

Summary Cyclic variations in resistance to ozone-inactivation were observed during the cell cycle of a haploid strain of Saccharomyces cerevisiae. The lag and G₁ phases showed the highest sensitivity to ozone whereas there was a progressive increase in resistance from the S to G₂ phases as the proportion of dividing cells increased. A related series of S. cerevisiae varying in ploidy from haploid to tetraploid was exposed to ozone. The results demonstrated clearly an enhancement of ozone resistance directly related to the ploidy of the cells.Taken together these data suggest that resistance to ozone is dependent upon the action of an efficient repair mechanism. Similarities and differences between the biological action of ozone and ionizing radiations in S. cerevisiae are discussed.     

(-)-Epigallocatechin gallate regulates CD3-mediated T cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase

The zeta chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 micromol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cgamma1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.     

Proneural bHLH neurogenin 2 differentially regulates Nurr1-induced dopamine neuron differentiation in rat and mouse neural precursor cells in vitro

Roles of Nurr1 and neurogenin 2 (Ngn2) have been shown in midbrain dopamine (DA) neuron development. We present here rat and mouse species-dependent differences of Nurr1 and Ngn2 actions in DA neuron differentiation. Nurr1 exogene expression caused an efficient generation of tyrosine hydroxylase (TH)-positive DA cells from rat neural precursor cells (NPCs). Nurr1-induced TH+ cell yields were low and highly variable depending on the origins of NPCs in mouse cultures. Coexpression of Ngn2 repressed Nurr1-induced generation of TH+ cells in rat cultures. In clear contrast, a robust enhancement in Nurr1-induced DA cell yields was observed in mouse NPCs by Ngn2. These findings imply that DA neurons may develop differently in the midbrains of these two species.