3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.     

VanB-vanA incongruent VRE isolated from animals and humans in 1999

16 chicken isolates and four clinical isolates of VanB-vanA incongruent vancomycinresistant Enterococcus faecium strains without vanS were isolated in 1999. Pulsed-field gel electrophoresis revealed only a peripheral relationship between the chicken isolates and clinical isolates, but suggested clonal spread in the chicken isolates.     

Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes

A robust and fast DNA chip method was developed in order to detect the various beta-lactam antibiotic-resistance genes in one slide. These genes included PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY, and AmpC. beta-lactam antibiotic-resistance genes were labeled with a fluorescent nucleotide by a multiplex polymerase chain reaction using a mixture of specific primer sets for each gene. This labeled target was hybridized with a DNA chip that contained the spots of the specific probe DNAs for each beta-lactam antibiotic-resistance gene. This technique made it possible to detect the specific resistance gene, even in a single bacterium.     

Neutrophil-to-eosinophil ratio as a biomarker for clinical outcomes in advanced stage melanoma patients treated with anti-PD-1 therapy

Neutrophil-to-lymphocyte ratios (NLR) and eosinophil counts are associated with improved survival in melanoma patients treated with immune checkpoint inhibitors, but no study has investigated neutrophil-to-eosinophil ratios (NER) as a predictive indicator in this population. In this retrospective study evaluating anti-PD-1 treated patients with advanced melanoma, progression-free survival (PFS), overall survival (OS), objective response rates (ORR), and risk of high-grade (grade ≥3) immune-related adverse events (irAEs) were compared between groups defined by median pretreatment NLR and NER as well as median NLR and NER at 1-month post-treatment. Lower baseline NLR and NER were associated with improved OS [HR: 0.504, 95% CI: 0.328–0.773, p = .002 and HR: 0.442, 95% CI: 0.288–0.681, p < .001, respectively] on univariate testing. After accounting for multiple covariates, our multivariate analysis found that lower pretreatment NER was associated with better ORR (by irRECIST) (OR: 2.199, 95% CI: 1.071–4.582, p = .033) and improved OS (HR: 0.480, 95% CI: 0.296–0.777, p = .003). Baseline NLR, 1-month NLR, and 1-month NER were not associated with ORR, PFS, or OS outcomes; but 1-month NER correlated with lower risk of grade ≥3 irAEs (OR: 0.392, 95% CI: 0.165–0.895, p = .029). Our findings suggest baseline NER merits additional investigation as a novel prognostic marker for advanced melanoma patients receiving anti-PD-1-based regimens.     

Analysis of the expression of microtubule plus-end tracking proteins (+TIPs) during Xenopus laevis embryogenesis

Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis.     

DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.     

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.     

Effect of Weissella confusa Strain PL9001 on the Adherence and Growth of Helicobacter pylori

Viable and nonviable Weissella confusa strain PL9001 inhibited the binding of Helicobacter pylori to human gastric-cell line MKN-45 cells by more than 90%. Spent culture supernatant of PL9001 rapidly decreased the viability of H. pylori, rupturing cell walls. The results suggest that PL9001 is a probiotic that can reduce the infectivity and persistence of H. pylori.     

Microarray-based expression analysis of human osteoblast-like cell response to anodized titanium surface

An anodized surface significantly enhanced the adhesion of human osteoblast-like MG-63 cells to titanium. Using cDNA microarray analysis, five genes were differentially expressed while the rest remained unaltered. The results demonstrated that the anodized surface enhances cellular adhesion without significantly affecting the pattern of gene expression.