Rapid Mass Spectrometry Imaging to Assess the Biochemical Profile of Pituitary Tissue for Potential Intraoperative Usage.

Pituitary adenomas are relatively common intracranial neoplasms that are frequently treated with surgical resection. Rapid visualization of pituitary tissue remains a challenge as current techniques either produce little to no information on hormone-secreting function or are too slow to practically aid in intraoperative or even perioperative decision-making. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) represents a powerful method by which molecular maps of tissue samples can be created, yielding a two-dimensional representation of the expression patterns of small molecules and proteins from biologic samples. In this chapter, we review the use of MALDI MSI, its application to the characterization of the pituitary gland, and its potential applications for guiding the management of pituitary adenomas.

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma.

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.

Regulation of heme oxygenase 1 expression by miR-27b with stem cell therapy for liver regeneration in rats.

Adipose-derived mesenchymal stem cells (ASCs) have been considered to be attractive and readily available adult mesenchymal stem cells (MSCs) and are becoming increasingly popular for use in regenerating cell therapy. However, recent evidence attributed a fibrotic potential to MSCs which differentiated into myofibroblasts with highly increased α-smooth muscle actin (α-SMA) expression while transplanted into an injured/regenerating liver in mice. In this study, we studied the role of miR-27b in ASCs and their regenerative potential after partial liver resection in rats. ASCs transfected with control siRNA or miR-27b were intravenously injected into autologous rats undergoing 70% partial hepatectomy (PH). Our data showed that the regenerative capacities of ASCs with overexpressed miR-27b were significantly higher compared with control ASCs. However, the enhanced regeneration, hepatic differentiation, and suppressed liver inflammation, as well as fibrotic activity, were significantly reverted by ZnPP coadministration (heme oxygenase-1 [HO-1] inhibitor) indicating an important role of HO-1 in the regenerating and cytoprotective activities of miR-27b-transfected ASCs. We demonstrated that administration of autologous ASCs overexpressed with miR-27b enhances rapid and early liver regeneration and, importantly, preserves function after PH. The ASCs with miR-27b overexpression might offer a viable therapeutic option to facilitate rapid recovery after liver resection.

miR-499A>G rs3746444 and miR-146aG>C expression and hepatocellular carcinoma risk in the Chinese population.

We conducted a case-control study of a possible association of miR-499A>G rs3746444 and miR-146aG>C rs2910164 with risk of hepatocellular carcinoma. Samples from 172 hepatocellular carcinoma patients and 185 cancer-free controls were collected from October 2008 to December 2011. PCR-RFLP analysis was performed to determine the polymorphisms in each individual. The MAFs of miR-146aG>C and miR-499A>G in controls were similar to that known from the SNP database, and frequencies of genotypes in controls were in line with Hardy-Weinberg equilibrium. We found that miR-499 AG was significantly associated with decreased risk for hepatocellular carcinoma when compared with miR-499 AA genotype (adjusted odds ration = 0.74, 95% confidence interval = 0.24-0.96). However, subjects carrying miR-146a GG had a non-significant 0.62-fold decreased risk of hepatocellular carcinoma. We did not find a significant association of miR-146aG>C rs2910164 and miR-499A>G rs3746444 polymorphisms with hepatocellular carcinoma risk in the Chinese population. Further investigations are warranted to clarify the relationship between miRNA polymorphisms and susceptibility to hepatocellular carcinoma risk in various ethnic populations.

Spinal meningeal diverticula in spontaneous intracranial hypotension: analysis of prevalence and myelographic appearance.

BACKGROUND AND PURPOSE: Spinal meningeal diverticula have been implicated in the pathogenesis of spontaneous intracranial hypotension and have been proposed as both diagnostic features of and therapeutic targets for the condition. We compared the prevalence and myelographic appearance of spinal diverticula in patients with SIH and healthy controls. MATERIALS AND METHODS: Patients satisfying the ICHD-2 criteria for SIH were retrospectively identified. CT myelograms of 19 patients with SIH were compared with CT myelograms of 18 control patients. Images were reviewed by 2 blinded neuroradiologists. The prevalence, morphology (round versus multilobulated), size, and location (cervical, upper thoracic, lower thoracic, or lumbar) of spinal meningeal diverticula were analyzed. RESULTS: There was no difference in the proportion of patients with diverticula in the SIH group compared with the control group (68% versus 44%, P = .14) or in the mean number of diverticula per patient (6.3 versus 2.2, P = .099). No difference was seen in the morphology (P = .95) or size (P = .71) of diverticula between groups. There was a difference between groups that just reached statistical significance (P = .050) in the location of the diverticula along the spinal axis, but substantial overlap was seen between groups for all spinal locations. CONCLUSIONS: Despite the well-established association between spinal meningeal diverticula and SIH, we found no difference in the prevalence or myelographic appearance of diverticula in patients with SIH compared with controls. Further investigation into the role of diverticula in the diagnosis and treatment of SIH is necessary.

The clinical differences between dengue and scrub typhus with acute respiratory failure in southern Taiwan.

BACKGROUND: For both dengue and scrub typhus, acute respiratory failure (ARF) is a serious complication. The present study was carried out in order to investigate the clinical courses and outcomes of adult dengue and scrub typhus patients with ARF, and to identify the clinical differences between adult dengue and scrub typhus patients with ARF. METHODS: We conducted a retrospective study of the serologically confirmed adult dengue or scrub typhus patients admitted between 1998 and 2008 at Kaohsiung Chang Gung Memorial Hospital. A total of 980 dengue and 102 scrub typhus adult patients were included in our study. RESULTS: Eighteen of the 980 adult dengue patients and 8 of the 102 adult scrub typhus patients had ARF. There were significant differences that existed for eschar (P = 0.001; dengue 0%; scrub 62.5%), cough (P = 0.016; dengue 55.6%; scrub typhus 100%), white blood cell (WBC) count [P = 0.026; dengue 7.40 ± 5.74; scrub typhus 11.84 ± 4.95 (×10(3)/µL)], platelet count [P = 0.008; dengue 42.2 ± 33.9; scrub typhus 104.1 ± 93.3 (×10(9)/L)], prothrombin time (PT) [P = 0.007; dengue 12.82 ± 1.36; scrub typhus 10.74 ± 0.98 (s)], activated partial thromboplastin time (APTT) [P = 0.002; dengue 50.81 ± 10.08; scrub typhus 37.44 ± 4.06 (s)], blood urea nitrogen (BUN) [P < 0.001; dengue 64.6 ± 43.2; scrub typhus 20.9 ± 9.1 (mg/dL)], creatinine [P < 0.001; dengue 3.77 ± 3.37; scrub typhus 1.05 ± 0.37 (mg/dL)], admission day (A-day) [P = 0.027; dengue 2.9 ± 1.3; scrub typhus 5.4 ± 2.6 (days)], and ventilator duration [P = 0.022; dengue 9.4 ± 14.0; scrub typhus 14.8 ± 10.4 (days)] between both groups. CONCLUSIONS: This study provides relatively rare data regarding the clinical differences between adult dengue and scrub typhus patients with ARF.

Effect of Ag underlayer on microstructures and perpendicular magnetic properties of CoPt nanocomposite thin films.

CoPt/Ag films were prepared by magnetron sputtering on glass substrates and subsequent annealing. The dependence of degree of ordering and magnetic properties on Ag film thickness and annealing conditions were investigated. It was found that the Ag underlayer played a dominant role in inducing the (001) texture of the CoPt film after annealing. CoPt films with a thickness about 20 nm and Ag underlayers with a thickness about 70 nm are easy to obtain a large degree of ordering and a perpendicular magnetic anisotropy after annealing at 700 degrees C for 30 min. CoPt/Ag films with out-of-plane coercivity (Hc (perpendicular)) in the range of 13.5-14.0 kOe and a out-of-plane squareness (S(perpendicular)) of 0.97 were obtained after annealing at 700 degrees C for 30 min. Ag underlayer is beneficial to enhance the Hc(perpendicular)and S(perpendicular) of CoPt film significantly. The degree of ordering and perpendicular magnetic properties of the CoPt films which deposited on Ag underlayer are larger than those of the single layer CoPt films.

A novel ultrahigh-resolution surface plasmon resonance biosensor with an Au nanocluster-embedded dielectric film.

The detection performance of conventional surface plasmon resonance (SPR) biosensors is limited to a 1 pg/mm(2) surface coverage of biomolecules, and consequently, such sensors struggle to detect the interaction of small molecules in low concentrations. The present study is attempted to propose the use of a novel SPR biosensor with Au nanoclusters embedded in a dielectric film to achieve a 10-fold improvement in the resolution performance. A co-sputtering method utilizing a multi-target sputtering system is used to fabricate the present dielectric films (SiO(2)) with embedded Au nanoclusters. It is shown that the sensitivity of the developed SPR biosensor can be improved by adjusting the size and volume fraction of the embedded Au nanoclusters in order to control the surface plasmon effect. The present gas detection and DNA hybridization experimental results confirm that the proposed Au nanocluster-enhanced SPR biosensor provides the potential to achieve an ultrahigh-resolution detection performance of approximately 0.1 pg/mm(2) surface coverage of biomolecules.

Modulation of nitric oxide bioavailability by erythrocytes.

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs.

Evidence for inhibition of low density lipoprotein oxidation and cholesterol accumulation by apolipoprotein H (beta2-glycoprotein I).

Low density lipoprotein (LDL) oxidation and lipid accumulation are thought to enhance the progression of atherosclerosis. Apolipoprotein H (apoH) has been implicated in the development of human atherosclerosis. However, the roles of apoH in the oxidative modification of LDL and cellular accumulation of lipid constituents remained uncharacterized. In this study, the level of plasma apoH was found to be significantly associated with the oxidative susceptibility of LDL in human subjects. Plasma levels of apoH were positively correlated with the lag time but negatively correlated with LDL oxidation rate in conjugated diene formation. By using a J774 A.1 macrophage culture system, we found that apoH could not only inhibit the formation of conjugated diene and thiobarbituric acid-reactive substances, but also reduce the electrophoretic mobility of oxidized LDL. Furthermore, apoH decreased cellular accumulation of cholesterol via a reduction in cholesterol influx and an increase in cholesterol efflux. This is the first demonstration that apoH appears to have "antioxidant"-like effects on LDL oxidation. The results also suggest that apoH can inhibit the translocation of cholesterol from extracellular pools to macrophages, suggesting that apoH may play an important role in the prevention of atherosclerosis.

Erythrocyte consumption of nitric oxide: competition experiment and model analysis.

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.

Liquid-phase parallel synthesis of ureas.

An efficient and general liquid-phase method has been developed for the synthesis of a piperazine containing urea library. Reactions of the polymer bound carbamoyl chloride with primary or secondary amines afford ureas at ambient temperature. Desired compounds are liberated from the polymer support under mild conditions in high yields and high purity by simple precipitation and washings.

Age-associated changes in interferon-gamma and interleukin-4 secretion by purified human CD4+ and CD8+ T cells.

Aging is associated with a decline in immune function. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-gamma after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-gamma-producing T helper 1 and T cytotoxic 1 cells.

Erythrocytes possess an intrinsic barrier to nitric oxide consumption.

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.

Intravascular flow decreases erythrocyte consumption of nitric oxide.

Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), k(Hb)= 3-5 x 10(7) M(-1).s(-1), suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular flow does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular flow reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 microM) completely inhibits NO-mediated dilation with or without intravascular flow. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular flow does not affect the reaction rate between NO and free Hb in the lumen, because the latter forms a homogeneous solution and is not subject to the hydrodynamic separation. However, intravascular flow only partially contributes to the reduced consumption of NO by RBCs, because without the flow, the NO consumption by RBCs is already about 3 orders of magnitude slower than free Hb.

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing.

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.

Lipopolysaccharide activates endothelial nitric oxide synthase through protein tyrosine kinase.

Vascular endothelial cell injury or activation by lipopolysaccharide (LPS) plays an important role in the pathogenesis of endotoxin shock. However, the effect of LPS on NO production from vascular endothelial cells (ECs) is incompletely understood. In this study, bovine coronary venular ECs were treated with LPS and the release of NO and expression of the endothelial NO synthase (ecNOS) were examined. We found that the ecNOS activity is transiently enhanced by LPS within the time scale of about 10 h due to the interplay between two LPS-induced mechanisms. Within the first 10 h of LPS treatment, the specific activity of ecNOS is increased by a post-translational modification mediated through a protein tyrosine kinase cascade. After about 10 h of treatment, however, LPS destabilizes the transcript of ecNOS and thus decreased the expression level and total activity.

Clinical application of intratumoral blood flow study in patients with endometrial carcinoma.

BACKGROUND: The objective of this study was to evaluate the correlation between intratumoral blood flow as assessed by color Doppler ultrasound with stage, tumor grade, depth of invasion, and lymph node metastasis in endometrial carcinoma and determine its clinical usefulness. METHODS: Sixty-six patients with endometrial carcinoma were enrolled. All patients received surgical treatment. Transvaginal color Doppler ultrasound was performed before surgery to detect the arterial blood flow signals within the tumors and the lowest resistance index (RI) was recorded. Formalin fixed, paraffin embedded pathology slides were reviewed by a senior pathologist to evaluate the histologic grading, tumor size, depth of myometrial invasion, and presence of lymph node metastasis. RESULTS: Intratumoral RI correlated well with surgical staging, histologic grading, the depth of myometrial invasion, and the presence of lymph node metastasis. Significantly lower RI was noted in tumors of advanced stage (> than International Federation of Obstetrics and Gynecology [FIGO] Stage I) (0.38 +/- 0.09 vs. 0.54 +/- 0.11; P < 0.001), tumors with higher histologic grade (Grade 3) (0.36 +/- 0.08 vs. 0.53 +/- 0.11; P < 0.001), tumors with deep myometrial invasion (> 50% myometrial thickness) (0.38 +/- 0.07 vs. 0.54 +/- 0.11; P < 0.001), and tumors with lymph node metastasis (0.34 +/- 0.07 vs. 0.52 +/- 0.11; P < 0.001) compared with tumors with Stage I, Grade 1/2 histology, no or superficial myometrial invasion, and absence of lymph node metastasis, respectively. CONCLUSIONS: Intratumoral blood flow analysis assessed by color Doppler ultrasound correlates well with surgical stage, tumor grade, myometrial invasion, and lymph node metastasis in patients with endometrial carcinoma. Assessment of tumor angiogenesis using color Doppler ultrasound provides useful information for the preoperative prediction regarding stage, histologic grade, depth of myometrial invasion, and presence of lymph node metastasis in patients with endometrial carcinoma. The authors believe routine pelvic lymph node dissection should be performed for those patients whose lowest RI values of intratumoral blood flow are < or = 0.4 because those patients are at very high risk for pelvic lymph node involvement.