Limited efficacy of specific HDAC6 inhibition in urothelial cancer cells

Epigenetic modifiers such as histone deacetylases (HDACs) have come into focus as novel drug targets for cancer therapy due to their functional role in tumor progression. Since common pan-HDAC inhibitors have adverse side effects and minor anti-cancer activity against solid tumors, enzyme-specific inhibitors were developed. HDAC6 is especially well-suited for specific inhibition due to its unique domain structure and mode of action and has been suggested to provide an exceptionally suitable target for cancer therapy. However, expression and function of HDACs have been insufficiently studied in urothelial cancers (UC), a disease urgently requiring new therapeutic approaches. The present study sought to evaluate HDAC6 as a target for treatment of urothelial cancers with enzyme-specific inhibitors. We observed moderate HDAC6 overexpression in urothelial cancer tissues and a broad range of expression in urothelial cancer cell lines. In the cell lines Tubacin was the most potent inhibitor, compared with Tubastatin and ST-80, but still active only at high micromolar concentrations. HDAC6 expression levels correlated poorly with sensitivity to enzyme inhibition. Combined treatments with heat shock, HSP90 inhibition by 17-AAG, proteasome inhibition by bortezomib, or DNA-damaging agents did not result in significant synergistic effects. Experiments with siRNA-mediated knockdown further underlined that urothelial cancer cells do not critically depend on HDAC6 expression for survival.     

Butyrate Induces Glutathione S-Transferase in Human Colon Cells and Protects From Genetic Damage by 4-Hydroxy-2-Nonenal

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 μM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24- 72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.     

Parahydrogen‐induced polarization of carboxylic acids: a pilot study of valproic acid and related structures

Parahydrogen‐induced polarization (PHIP) is a promising new tool for medical applications of MR, including MRI. The PHIP technique can be used to transfer high non‐Boltzmann polarization, derived from parahydrogen, to isotopes with a low natural abundance or low gyromagnetic ratio (e.g. ¹³C), thus improving the signal‐to‐noise ratio by several orders of magnitude. A few molecules acting as metabolic sensors have already been hyperpolarized with PHIP, but the direct hyperpolarization of drugs used to treat neurological disorders has not been accomplished until now. Here, we report on the first successful hyperpolarization of valproate (valproic acid, VPA), an important and commonly used antiepileptic drug. Hyperpolarization was confirmed by detecting the corresponding signal patterns in the ¹H NMR spectrum. To identify the optimal experimental conditions for the conversion of an appropriate VPA precursor, structurally related molecules with different side chains were analyzed in different solvents using various catalytic systems. The presented results include hyperpolarized ¹³C NMR spectra and proton images of related systems, confirming their applicability for MR studies. PHIP‐based polarization enhancement may provide a new MR technique to monitor the spatial distribution of valproate in brain tissue and to analyze metabolic pathways after valproate administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Step-function luminopsins for bimodal prolonged neuromodulation

Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non-invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto-chemogenetic probes termed luminopsins by fusing light-sensing opsins with light-emitting luciferases. In this report, we incorporated Chlamydomonas channelrhodopsin 2 with step-function mutations as the opsin moiety in the new luminopsin fusion protein termed step-function luminopsin (SFLMO). Bioluminescence-induced photocurrent lasted longer than the bioluminescence signal due to very slow deactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals.     

Making Meals in Small Seasonal Restaurants

Since restaurateurs can benefit by analyzing the production of meals, particularly with the dominant framework for meal experiences, the five aspects meal model (FAMM), this study examined FAMM’s relevance as an analytical tool for understanding meal production via field observations and interviews in eight small restaurants in a rural destination in Sweden. Results showed that FAMM’s aspect of the management control system and the factor of time are critical to the entire meal production process in restaurants. This article closes with a discussion of FAMM’s usefulness as a qualitative checklist for restaurateurs.     

Liver dysfunction in Pennsylvania's multitransfused hemophiliacs

Transaminase values [alanine amino transferase (ALT) and aspartate amino transferase (AST)] and markers for hepatitis B were serially determined in 558 hemophiliacs exposed to blood products. Hepatitis B surface antigen (HB_sAg) persistent for over 12 months was present in 6% of the patients. Antibody to hepatitis B surface antigen (anti-HBs) was noted in 90% of the 259 patients treated with factor VIII or IX concentrates but in only 49% of the 43 patients treated with fresh frozen plasma (FFP) or cryoprecipitate. Persistently abnormal transaminase values were noted in 31% of the patients treated with commercial concentrates but in only one (2%) of the patients exposed to cryoprecipitate or FFP. This difference continued even when the two groups of patients were matched for the amount of blood products, up to 50, 000 units, which they had received in the study period. In the concentrate-treated patients, no correlation could be found between transaminase values and the number of units of factor VIII or IX they had received during the six years of the study (1973–1978).Supported in part by the Pennsylvania Hemophilia Centers and the Pennsylvania State-Wide Hemophilia Program.     

Mesenteric pulsatility index analysis predicts response to azathioprine in patients with Crohn's disease

OBJECTIVE: Mesenteric blood flow measurement has been found to predict relapse after steroid-induced remission in patients with Crohn's disease (CD) and ulcerative colitis (UC). Therefore, we assessed prospectively the possible relationship between changes in mesenteric blood flow and prognosis in chronically active patients with need of immunosuppressive therapy with azathioprine (AZA) or 6-mercaptopurine (6-MP). METHODS: Doppler ultrasound (DUS) measurements of the pulsatility index (PI) of the superior mesenteric artery (SMA) and inferior mesenteric artery (IMA) were performed in 52 patients with chronically active inflammatory bowel disease (CD 31 patients; UC 21 patients) before beginning therapy with AZA/6-MP (US1) and during clinical remission (CD activity index <150, Truelove index score I) (US2). Patients were weaned from concomitant therapy with corticosteroids as soon as possible and were followed up for 12 months. RESULTS: After 1 year, 16 patients with CD (51.6%) and 13 patients with UC (61.9%) were in remission, whereas 23 patients had recurrent disease or had undergone surgery. A decreased SMA PI at US2 predicted clinical relapse in all patients with CD [100%; P < 0.001; mean (+/-SD) 77 +/- 67 d after US1], but only 4 of 8 patients (50%; difference not significant; mean 84 +/- 75 d after US1) with UC. Conversely, an increase of SMA PI was associated with sustained remission in the majority of CD patients (12/16 patients; 75%; P < 0.002), but in only 7 of 13 patients (54%) with UC. Flow measurements in the IMA and postprandial values for both arteries were less reliable. CONCLUSION: Repeated DUS measurements of the SMA PI predict response to AZA/6-MP in patients with chronic active CD.     

Deciding on Gender in Children with Intersex Conditions

Biologic factors such as genetic and hormonal influences contribute to gender identity, gender role behavior, and sexual orientation in humans, but this relationship is considerably modified by psychologic, social, and cultural factors. The recognition of biologically determined conditions leading to incongruity of genetically determined sex, somatic phenotype, and gender identity has led to growing interest in gender role development and gender identity in individuals with intersex conditions. Sex assignment of children with ambiguous genitalia remains a difficult decision for the families involved and subject to controversial discussion among professionals and self-help groups. Although systematic empirical data on outcomes of functioning and health-related quality of life are sparse, anecdotal evidence from case series and individual patients about their experiences in healthcare suggests traumatic experiences in some. This article reviews the earlier 'optimal gender policy' as well as the more recent 'full consent policy' and reviews published data on both surgical and psychosocial outcomes. The professional debate on deciding on sex assignment in children with intersex conditions is embedded in a much wider public discourse on gender as a social construction. Given that the empirical basis of our knowledge of the causes, treatment options, long-term outcomes, and patient preferences is insufficient, we suggest preliminary recommendations based on clinical experience, study of the literature, and interviews with affected individuals.