A small-scale, rolled-membrane microfluidic artificial lung designed towards future large area manufacturing.

Artificial lungs have been used in the clinic for multiple decades to supplement patient pulmonary function. Recently, small-scale microfluidic artificial lungs (µAL) have been demonstrated with large surface area to blood volume ratios, biomimetic blood flow paths, and pressure drops compatible with pumpless operation. Initial small-scale microfluidic devices with blood flow rates in the µl/min to ml/min range have exhibited excellent gas transfer efficiencies; however, current manufacturing techniques may not be suitable for scaling up to human applications. Here, we present a new manufacturing technology for a microfluidic artificial lung in which the structure is assembled via a continuous "rolling" and bonding procedure from a single, patterned layer of polydimethyl siloxane (PDMS). This method is demonstrated in a small-scale four-layer device, but is expected to easily scale to larger area devices. The presented devices have a biomimetic branching blood flow network, 10 µm tall artificial capillaries, and a 66 µm thick gas transfer membrane. Gas transfer efficiency in blood was evaluated over a range of blood flow rates (0.1-1.25 ml/min) for two different sweep gases (pure O2, atmospheric air). The achieved gas transfer data closely follow predicted theoretical values for oxygenation and CO2 removal, while pressure drop is marginally higher than predicted. This work is the first step in developing a scalable method for creating large area microfluidic artificial lungs. Although designed for microfluidic artificial lungs, the presented technique is expected to result in the first manufacturing method capable of simply and easily creating large area microfluidic devices from PDMS.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues.

ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance.

Onset of the cancer mesenchymal program is closely associated with cancer malignancy and drug resistance. Among the different epithelial-mesenchymal transition (EMT)-associated transcriptional factors, ZEB1 has a key role in inducing the mesenchymal phenotypes and stem cell-like properties of different breast cancer cells. ARF6 and its effector AMAP1 are frequently overexpressed in breast cancer cells, and promote invasion, metastasis and drug resistance. EPB41L5 is induced during EMT, and mediates the disruption of E-cadherin-based cell-cell adhesion and the promotion of focal adhesion dynamics. Here we show that EPB41L5 is an integral component of the ARF6-based pathway, which is induced by ZEB1. We found that EPB41L5 is expressed at high levels in malignant breast cancer cells and binds to AMAP1. ZEB1 induced EPB41L5 both in cancer cells and normal cells. This relationship was recaptured with The Cancer Genome Atlas RNASeq data set, and correlated with the poor outcome of the patients. In contrast, diversified events, such as tumor growth factor ß1 stimulation, expression of SNAI1 and TP53 mutation, can each cause the induction of ZEB1 and EPB41L5, depending on the cellular context. Our results demonstrated that the ZEB1-EPB41L5 axis is at the core of the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance of significant populations of primary breast cancers, and is tightly correlated with the poor outcomes of patients.

Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group.

The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR-ABL-negative ALL. Patients aged 15-24 years (n=139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58-75%) and 73% (95% CI 64-80%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, P<0.001). These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR-ABL-negative ALL.

Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Interleukin-10 gene polymorphism reflects the severity of chronic immune thrombocytopenia in Japanese patients.

INTRODUCTION: T-helper cell type 1 (Th1) polarization of the immune response has been documented in patients with chronic immune thrombocytopenia (ITP). Interleukin (IL)-10 is the most important factor regulating Th1 and T-helper type 2 cytokine synthesis. This study evaluated the impact of IL-10 polymorphisms on both susceptibility to, and severity of, chronic ITP. METHODS: We analyzed -1082(G/A), -812(C/T), and -592(C/A) IL-10 polymorphisms in 90 patients with adult chronic ITP and 202 race- and sex-matched healthy controls. RESULTS: No significant differences in the genotype or haplotype frequencies were observed between the patient with chronic ITP and the control group. However, more patients with the -592AA genotype showed a severe thrombocytopenic state (platelet count <10 x 109/l) than those with the -592CC/CA genotypes (44.1%vs. 19.6%, P = 0.01). Furthermore, more patients with the ATA/ATA haplotype showed a severe thrombocytopenic state than those without the ATA/ATA haplotype (44.1%vs. 19.6%, P = 0.01). CONCLUSION: According to our data, patients with low producer type of IL-10 polymorphisms have more severe thrombocytopenia, suggesting that IL-10 gene polymorphisms may reflect the severity of ITP.

Visceral obesity is associated with the metabolic syndrome and elevated plasma retinol binding protein-4 level in obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) is related to the increased prevalence of cardiovascular disease and metabolic syndrome (MS). A novel adipokine, retinol binding protein-4 (RBP4), was reported to be associated with insulin resistance and the prevalence of type 2 diabetes. To examine whether plasma RBP4 is associated with insulin resistance and MS development in OSAS, we measured plasma RBP4 levels in 181 Japanese men (24 healthy controls and 40 mild, 64 moderate, and 53 severe OSAS) of whom 26 had mild glucose intolerance with HbA1c < or = 6.0%. After a full polysomnography, blood was collected between 06:00 and 07:00 AM. Plasma RBP4 levels in moderate/severe OSAS patients were higher than in control subjects. Plasma RBP4 was not correlated with apnea variables, HOMA-IR, or blood pressure. However, it was positively correlated with visceral fat areas and plasma triglyceride levels. The prevalence of MS was higher in severe OSAS patients than in mild/moderate OSAS and control subjects. Plasma RBP4 was higher in OSAS patients with MS than in those without MS. This study indicates that plasma RBP4 is associated with dyslipidemia, but not with insulin resistance, glucose intolerance, or hypertension in patients with OSAS. Visceral obesity may play key roles in increasing the plasma RBP4 level and MS development in OSAS.

Clinical significance of Th1/Th2 ratio in patients with myelodysplastic syndrome.

In this study, we attempted to evaluate the clinical significance of T helper 1 (Th1)/T helper 2 (Th2) ratio in patients with myelodysplastic syndrome (MDS), five refractory anaemia (RA), four refractory anaemia with ringed sideroblasts (RARS), 31 refractory cytopenia with multilineage dysplasia (RCMD), nine refractory anaemia with excess blast-1 (RAEB-1) and seven refractory anaemia with excess blast-2 (RAEB-2). Intracellular interleukin-4 (Th2 cytokine) and interferon-gamma (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin using flow cytometry. Mean Th1/Th2 ratios in each MDS group were as follows: RA/RARS, 8.8 (95% CI, 5.8-11.8), RCMD, 14.7 (95% CI, 9.5-19.9), RAEB-1, 10.6 (95% CI, 4.6-16.6), RAEB-2, 12.8 (95% CI, 3.0-22.7) and control 12.8 (95% CI, 9.6-16.1). There were no significant differences in Th1/Th2 ratio in the RA/RARS, RCMD, RAEB-1 and RAEB-2 subgroups when compared to controls. Because Th1/Th2 ratio in the RCMD group was widely distributed, we divided RCMD patients according to Th1/Th2 ratio into three groups (low, normal and high Th1/Th2 groups). There were no differences in severity of cytopenia among the three above groups. However, the percentage of CD8 cells in the low Th1/Th2 group was significantly lower than those in the high group (P < 0.01). These data suggest that Th1/Th2 imbalance induces CD4/CD8 imbalance, and serves as a marker of the biological interplay in immune regulation.

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen.

Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.

Comparative uterine gene expression analysis after dioxin and estradiol administration.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many organisms. TCDD exposure is known to be associated with abnormal development, hepatotoxicity and endocrine effects. It has also been reported to have antiestrogenic activity in addition to estrogenic activity. In order to clarify the effects of TCDD in the uterus, we evaluated the patterns of gene expression after TCDD and estradiol administration. Of the 10 000 arrayed genes, only a few were affected by both estradiol and TCDD. Although the subset of genes that responded to estrogen was also activated by TCDD, the response to TCDD was more limited than that observed in response to estradiol. Therefore, according to our analysis of gene expression patterns, TCDD had partial and weak estrogenic activity in the uterus.

Tissue-specific estrogenic and non-estrogenic effects of a xenoestrogen, nonylphenol.

Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparable with the uterotrophic assay, it indicated that analysis of gene expression profiles can predict the estrogenic activities of chemicals. In contrast to the similar effects of nonylphenol and estradiol observed in the uterus, in the liver, gene expression was more markedly affected by nonylphenol than by estradiol. This indicated that, in the liver, nonylphenol could activate another set of genes that are distinct from estrogen-responsive genes. These results indicated that nonylphenol has very similar effects to estradiol on gene expression in uterine but not in liver tissue, indicating that tissue-specific effects should be considered in order to elucidate the distinct effects of alkylphenols.

Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol.

Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments.

Despite recent progress in sequencing the complete genome of rice ( Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.

Differentially expressed Maf family transcription factors, c-Maf and MafA, activate glucagon and insulin gene expression in pancreatic islet alpha- and beta-cells.

A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activation of the glucagon promoter by c-Maf was more efficient than by the other alpha-cell-enriched transcription factors, Cdx2, Pax6, and Isl-1. Furthermore, inhibition of c-Maf expression in alphaTC1 cells by specific short hairpin RNA resulted in marked reduction of the glucagon promoter activity. Thus, c-Maf and MafA are differentially expressed in alpha- and beta-cells where they regulate glucagon and insulin gene expression, respectively.

Durable remission induced by rituximab-containing chemotherapy in a patient with primary refractory Burkitt's lymphoma.

We describe a 65-year-old man diagnosed with Burkitt's lymphoma arising from the intestine. The tumor cells had a mature B-cell immunophenotype and rearrangement of the c-myc gene. The patient was treated with intensive multiagent chemotherapy. After four courses of chemotherapy, an ileus developed due to a residual abdominal disease. We administered rituximab in combination with the same chemotherapy regimen. A dramatic clinical improvement was observed and abnormal uptake by 18F-fluorodeoxyglucose positron emission tomography disappeared. The patient experienced complete remission for 1 year. This encouraging result indicates that rituximab might be an important treatment choice in management of Burkitt's lymphoma.

Isolation and characterization of an alternatively spliced variant of transcription factor Islet-1.

The LIM homeodomain protein Islet-1 (Isl1), one of the earliest markers for motor neuron differentiation, is also expressed in all classes of islet cells in the pancreas. Isl1 is known to bind and regulate the promoters of the insulin, glucagon and somatostatin genes. In this study, we describe isolation of a novel isl1 cDNA species from the mouse islet beta cell line betaTC6, which arose from the utilization of an alternative splicing acceptor site in the fifth exon. This shorter cDNA encodes an Isl1 isoform (Isl1-beta) lacking the carboxy-terminal 23 amino acids of the previously reported product Isl1-alpha. Although the level of isl1-beta mRNA is much lower than that of isl1-alpha, isl1-beta is preferentially expressed in murine insulinoma cell lines but not in glucagonoma cell line. Upon transient transfection, both Isl1-alpha and Isl1-beta accumulate in the nuclei of murine insulinoma cells. We found that Isl1-beta is a relatively more potent transcriptional activator of the insulin promoter than Isl1-alpha and that the Isl1-alpha isoform undergoes phosphorylation. Therefore, the transcriptional activity of Isl1 is potentially regulated by the alternative splicing of its mRNA and by phosphorylation.

Similarities and differences in uterine gene expression patterns caused by treatment with physiological and non-physiological estrogens.

Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.

Analysis of temporal changes in the expression of estrogen-regulated genes in the uterus.

In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.