Continuous roller nanoimprinting: next generation lithography.

Nanoimprint lithography (NIL) is a cost-effective and high-throughput technique for replicating nanoscale structures that does not require expensive light sources for advanced photolithography equipment. NIL overcomes the limitations of light diffraction or beam scattering in traditional photolithography and is suitable for replicating nanoscale structures with high resolution. Roller nanoimprint lithography (R-NIL) is the most common NIL technique benefiting large-scale, continuous, and efficient industrial production. In the past two decades, a range of R-NIL equipment has emerged to meet the industrial needs for applications including biomedical devices, semiconductors, flexible electronics, optical films, and interface functional materials. R-NIL equipment has a simple and compact design, which allows multiple units to be clustered together for increased productivity. These units include transmission control, resist coating, resist curing, and imprinting. This critical review summarizes the hitherto R-NIL processes, their typical technical problems, and corresponding solutions and gives guidelines for developing advanced R-NIL equipment.

Early Synaptic Alterations and Selective Adhesion Signaling in Hippocampal Dendritic Zones Following Organophosphate Exposure.

Organophosphates account for many of the world's deadliest poisons. They inhibit acetylcholinesterase causing cholinergic crises that lead to seizures and death, while survivors commonly experience long-term neurological problems. Here, we treated brain explants with the organophosphate compound paraoxon and uncovered a unique mechanism of neurotoxicity. Paraoxon-exposed hippocampal slice cultures exhibited progressive declines in synaptophysin, synapsin II, and PSD-95, whereas reduction in GluR1 was slower and NeuN and Nissl staining showed no indications of neuronal damage. The distinctive synaptotoxicity was observed in dendritic zones of CA1 and dentate gyrus. Interestingly, declines in synapsin II dendritic labeling correlated with increased staining for ß1 integrin, a component of adhesion receptors that regulate synapse maintenance and plasticity. The paraoxon-induced ß1 integrin response was targeted to synapses, and the two-fold increase in ß1 integrin was selective as other synaptic adhesion molecules were unchanged. Additionally, ß1 integrin-cofilin signaling was triggered by the exposure and correlations were found between the extent of synaptic decline and the level of ß1 integrin responses. These findings identified organophosphate-mediated early and lasting synaptotoxicity which can explain delayed neurological dysfunction later in life. They also suggest that the interplay between synaptotoxic events and compensatory adhesion responses influences neuronal fate in exposed individuals.

Altered functionality of anti-bacterial antibodies in patients with chronic hepatitis C virus infection.

BACKGROUND: Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease. METHODS: The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined. RESULTS: Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure. CONCLUSIONS: Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.

Interleukin-6 and oncostatin M are elevated in liver disease in conjunction with candidate hepatocellular carcinoma biomarker GP73.

The Golgi phosphoprotein GP73 is elevated in the circulation of individuals with a diagnosis of hepatocellular carcinoma. Its usefulness as a biomarker of HCC is questioned, since it has also been reported to be elevated in the circulation of people with liver cirrhosis. Regulation of GP73 by inflammatory cytokines is therefore of interest. The interleukin-6 (IL-6) family cytokines were tested for effects on GP73 mRNA and/or protein levels in human hepatoblastoma HepG2 cells. Levels of GP73 mRNA and protein were up-regulated in HepG2 cells following treatment with either proinflammatory cytokine IL-6 or the related cytokine oncostatin M (OSM). Induction required the shared receptor subunit gp130, and correlated with increased tyrosine phosphorylation of STAT3. Maximal cytokine-mediated induction was not observed in the presence of protein synthesis inhibitor cycloheximide, suggesting additional regulatory factors play an important role. ELISA measurement of GP73 and IL-6 levels in the sera of patients with pre-malignant liver disease revealed a significant correlation between circulating levels of the two proteins. Similarly, a sensitive ELISA assay was developed to measure circulating OSM. OSM levels were elevated 6-7 fold in sera from patients with either cirrhosis or HCC relative to controls without liver disease. Although there was an association between levels of GP73 and OSM in serum from people with liver cirrhosis, there was not a statistically significant correlation in HCC, suggesting that the role of the cytokines in determining circulating levels may be complex. To our knowledge, this is the first report of OSM elevation being associated with liver disease.

Analysis of GP73 in patients with HCC as a function of anti-cancer treatment.

In this study, we examined the level of Golgi protein 73 (GP73) in the serum of 9 patients as a function of anti-liver cancer treatment. Although the numbers are small, a clear trend was observed. Patients who remained tumor free (up to 6 years post-treatment) showed reductions in GP73 at the first time point available post-treatment. In contrast, patients who had high levels GP73 post treatment all had re-occurrence within a 5 year period. These data are preliminary but dramatically imply that this marker may have value in the monitoring of HCC patients and may be elevated even when small, undetectable tumors are present.

Novel fucosylated biomarkers for the early detection of hepatocellular carcinoma.

Changes in glycosylation, most notably fucosylation, have been associated with the development of hepatocellular carcinoma (HCC). In this report, the levels of fucosylated kininogen (Fc-Kin) and fucosylated alpha-1-antitrypsin were analyzed individually and in combination with the currently used marker, alpha-fetoprotein, and a previously identified biomarker, Golgi protein 73 (GP73), for the ability to distinguish between a diagnosis of cirrhosis and HCC. This analysis was done on serum from 113 patients with cirrhosis and 164 serum samples from patients with cirrhosis plus HCC. The levels of Fc-Kin and fucosylated alpha-1-antitrypsin were significantly higher in patients with HCC compared with those with cirrhosis (P < 0.0001). Greatest performance was achieved through the combination of Fc-Kin, alpha-fetoprotein, and GP73, giving an optimal sensitivity of 95%, a specificity of 70%, and an area under the receiver operating characteristic of 0.94. In conclusion, the altered glycosylation of serum glycoproteins can act as potential biomarkers of primary HCC when used independently or in combination with other markers of HCC.

Identification and development of fucosylated glycoproteins as biomarkers of primary hepatocellular carcinoma.

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, we have previously reported changes in N-linked glycosylation that occur with the development of hepatocellular carcinoma (HCC) and, through the use of glycoproteomics, identified many of those proteins containing altered glycan structures. To advance these studies and further explore the glycoproteome, we performed N-linked glycan analysis from serum samples depleted of the major acute phase proteins, followed by targeted lectin extraction of those proteins containing changes in glycosylation. Using this method, changes in glycosylation, specifically increased amounts of core and outer arm fucosylation, were observed in the depleted samples. The identities of those proteins containing core and outer arm fucose were identified in the serum of patients with HCC. The usefulness of some of these proteins in the diagnosis of HCC was determined through the analysis of over 300 patient samples using a high-throughput plate based approach. Greatest performance was achieved with fucosylated hemopexin, which had an AUROC of 0.9515 with an optimal sensitivity of 92% and a specificity of 92%.

Planning during turmoil: credit challenges and healthcare finance. Interview by Carole J. Bolster.

The crisis in the financial markets is having a major impact on hospitals' ability to access capital. Providers are seeking longer-term fixed-rate debt rather than shortterm debt. Hospital management teams and their boards need to understand the upside and downside of variable-rate debt and interest rate derivatives.

Human immunodeficiency virus-related microbial translocation and progression of hepatitis C.

BACKGROUND & AIMS: Human immunodeficiency virus (HIV)-1 infection has been associated with enhanced microbial translocation, and microbial translocation is a mechanism through which alcohol and some enteric conditions cause liver disease. We hypothesized that HIV promotes liver disease by enhancing microbial translocation. METHODS: We studied human cohorts in which hepatitis C virus (HCV) and HIV outcomes were carefully characterized. RESULTS: HIV-related CD4(+) lymphocyte depletion was strongly associated with microbial translocation as indicated by elevated levels of circulating lipopolysaccharide (LPS), LPS-binding protein, soluble CD14, and fucose-binding lectin (AAL) reactive to immunoglobulin G specific for the alpha-galactose epitope and suppressed levels of endotoxin core antibodies (EndoCAb IgM) in HIV-infected subjects compared with the same persons before they had HIV infection and compared with HIV-uninfected subjects. The same measures of microbial translocation were strongly associated with HCV-related liver disease progression (cirrhosis), eg, LPS, odds ratio, 19.0 (P = .002); AAL, odds ratio, 27.8 (P < .0001); in addition, levels of LPS were elevated prior to recognition of cirrhosis. CONCLUSIONS: Microbial translocation may be a fundamental mechanism through which HIV accelerates progression of chronic liver disease.

Increased levels of galactose-deficient anti-Gal immunoglobulin G in the sera of hepatitis C virus-infected individuals with fibrosis and cirrhosis.

Hepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and liver cancer. Using comparative glycoproteomics, we identified a glycoprotein that is altered both in amount and in glycosylation as a function of liver fibrosis and cirrhosis. Specifically, this altered glycoprotein is an immunoglobulin G (IgG) molecule reactive to the heterophilic alpha-Gal epitope [Galalpha-1-3Galbeta1-(3)4GlcNAc-R]. While similar changes in glycosylation have been observed in several autoimmune diseases, the specific immunoglobulins and their antigen recognition profiles were not determined. Thus, we provide the first report identifying the specific antigenic recognition profile of an immunoglobulin molecule containing altered glycosylation as a function of liver disease. This change in glycosylation allowed increased reactivity with several fucose binding lectins and permitted the development of a plate-based assay to measure this change. Increased lectin reactivity was observed in 100% of the more than 200 individuals with stage III or greater fibrosis and appeared to be correlated with the degree of fibrosis. The reason for the alteration in the glycosylation of anti-Gal IgG is currently unclear but may be related to the natural history of the disease and may be useful in the noninvasive detection of fibrosis and cirrhosis.

Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma.

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). Chronic infection with the hepatitis B virus is associated with more than 55% of all cases of hepatocellular carcinoma. We show here that increased levels of core fucosylation can be observed via glycan analysis of total serum and are associated with the development of HCC. In a blinded study, the serum glycoproteins derived from people diagnosed with HBV induced liver cancer were found to possess a dramatically higher level of fucosylation. This change occurs on both immunoglobulin molecules and on other serum glycoproteins. Targeted glycoproteomic analysis was used to identify those glycoproteins that are hyperfucosylated in cancer. In total, 19 proteins were found to be hyperfucosylated in cancer. The potential of these proteins as biomarkers of cancer is discussed.

Endometriosis in adolescence: a long-term follow-up fecundability assessment.

OBJECTIVE: A long-term, follow-up study comparing mild and severe forms of endometriosis and their fecundability, on 28 women diagnosed with endometriosis in adolescence. METHODOLOGY: Twenty-eight patients were identified from a prospective cohort of 52 adolescents (ages 12 to 18 years) with operative diagnosis of endometriosis between July 1993 and December 1995. All patients presented with chronic pelvic pain unresponsive to conservative medical management. Diagnosis of pregnancy was made by sonographic identification of intrauterine pregnancy, positive serum human chorionic gonadotropin or pathological confirmation of products of conception. Patients were categorized as fertile or sub-fertile by having > 12 months of unprotected intercourse without conception. Follow-up was done for 8.6 years. RESULTS: Staging of endometriosis was performed according to the American Society for Reproductive Medicine standards. Stage I = 14.3%; Stage II = 39.3%; Stage III = 42.8%; Stage IV = 3.6%. Fecundability rates in each stage were statistically significant: Stage I (75%), Stage II (55%), Stage III (25%), Stage IV (0%) (p < .05). Rates of spontaneous abortion were not statistically significant. CONCLUSION: In our cohort, even at the earliest point in the natural life cycle of endometriosis there is an inverse relationship between stage of disease at diagnosis and fecundability.