Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration.

Sirtuin2 (Sirt2) with its NAD+-dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis of many diseases, thus making Sirt2 a promising target for pharmaceutical intervention. Herein, we present new high affinity Sirt2 selective Sirtuin-Rearranging Ligands (SirReals) that inhibit both Sirt2-dependent deacetylation and defatty-acylation in vitro and in cells. We show that simultaneous inhibition of both Sirt2 activities results in strongly reduced levels of the oncoprotein c-Myc and an inhibition of cancer cell migration. Furthermore, we describe the development of a NanoBRET-based assay for Sirt2, thereby providing a method to study cellular target engagement for Sirt2 in a straightforward and accurately quantifiable manner. Applying this assay, we could confirm cellular Sirt2 binding of our new Sirt2 inhibitors and correlate their anticancer effects with their cellular target engagement.

Polar Ocean Observations: A Critical Gap in the Observing System and Its Effect on Environmental Predictions From Hours to a Season.

There is a growing need for operational oceanographic predictions in both the Arctic and Antarctic polar regions. In the former, this is driven by a declining ice cover accompanied by an increase in maritime traffic and exploitation of marine resources. Oceanographic predictions in the Antarctic are also important, both to support Antarctic operations and also to help elucidate processes governing sea ice and ice shelf stability. However, a significant gap exists in the ocean observing system in polar regions, compared to most areas of the global ocean, hindering the reliability of ocean and sea ice forecasts. This gap can also be seen from the spread in ocean and sea ice reanalyses for polar regions which provide an estimate of their uncertainty. The reduced reliability of polar predictions may affect the quality of various applications including search and rescue, coupling with numerical weather and seasonal predictions, historical reconstructions (reanalysis), aquaculture and environmental management including environmental emergency response. Here, we outline the status of existing near-real time ocean observational efforts in polar regions, discuss gaps, and explore perspectives for the future. Specific recommendations include a renewed call for open access to data, especially real-time data, as a critical capability for improved sea ice and weather forecasting and other environmental prediction needs. Dedicated efforts are also needed to make use of additional observations made as part of the Year of Polar Prediction (YOPP; 2017-2019) to inform optimal observing system design. To provide a polar extension to the Argo network, it is recommended that a network of ice-borne sea ice and upper-ocean observing buoys be deployed and supported operationally in ice-covered areas together with autonomous profiling floats and gliders (potentially with ice detection capability) in seasonally ice covered seas. Finally, additional efforts to better measure and parameterize surface exchanges in polar regions are much needed to improve coupled environmental prediction.

Ocean variability and air-sea fluxes produced by atmospheric rivers.

Atmospheric rivers (ARs) cause heavy precipitation and flooding in the coastal areas of many mid-latitude continents, and thus the atmospheric processes associated with the AR have been intensively studied in recent years. However, AR-associated ocean variability and air-sea fluxes have received little attention because of the lack of high-resolution ocean data until recently. Here we demonstrate that typical ARs can generate strong upper ocean response and substantial air-sea fluxes using a high-resolution (1/12°) ocean reanalysis. AR events observed during the CalWater 2015 field campaign generate large-scale on-shore currents that hit the coast, generating strong narrow northward jets along the west coast of North America, in association with a substantial rise of sea level at the coast. In the open ocean, the AR generates prominent changes of mixed layer depth, especially south of 30°N due to the strong surface winds and air-sea heat fluxes. The prominent cooling of SST is observed only in the vicinity of AR upstream areas primarily due to the large latent heat flux. Using a long-term AR dataset, composite structure and variations of upper ocean and air-sea fluxes are presented, which are consistent with those found in the events during CalWater 2015.

Simultaneous Nitrite/Nitrate Imagery at Millimeter Scale through the Water-Sediment Interface.

The present study describes new procedures to obtain at millimeter resolution the spatial distribution of nitrite and nitrate in porewaters, combining diffusive equilibrium in thin films (DET), colorimetry and hyperspectral imagery. Nitrite distribution can be easily achieved by adapting the well-known colorimetric method from Griess (1879) and using a common flatbed scanner with a limit of detection about 1.7 µmol L(-1). Nitrate distribution can be obtained after reduction into nitrite by a vanadium chloride reagent. However, the concentration of vanadium chloride used in this protocol brings coloration with a wide spectral signature that creates interference only deconvolvable by imaging treatment from an entire visible spectrum for each pixel (spectral analysis). This can be achieved by hyperspectral imaging. The protocol retained in the present study allows obtaining a nitrite/nitrate image with micromolar limit of detection. The methods were applied in sediments from the Loire Estuary after different treatments and allowed to precisely describe two-dimensional millimeter features. The present technique adds to the combination of gel-colorimetry and hyperspectral imagery a very promising new application of wide interest for environmental issues in the context of early diagenesis and benthic fluxes.

LSD1 controls metastasis of androgen-independent prostate cancer cells through PXN and LPAR6.

Lysine-specific demethylase 1 (LSD1) was shown to control gene expression and cell proliferation of androgen-dependent prostate cancer (PCa) cells, whereas the role of LSD1 in androgen-independent metastatic prostate cancer remains elusive. Here, we show that depletion of LSD1 leads to increased migration and invasion of androgen-independent PCa cells. Transcriptome and cistrome analyses reveal that LSD1 regulates expression of lysophosphatidic acid receptor 6 (LPAR6) and cytoskeletal genes including the focal adhesion adaptor protein paxillin (PXN). Enhanced LPAR6 signalling upon LSD1 depletion promotes migration with concomitant phosphorylation of PXN. In mice LPAR6 overexpression enhances, whereas knockdown of LPAR6 abolishes metastasis of androgen-independent PCa cells. Taken together, we uncover a novel mechanism of how LSD1 controls metastasis and identify LPAR6 as a promising therapeutic target to treat metastatic prostate cancer.

Millimeter-scale alkalinity measurement in marine sediment using DET probes and colorimetric determination.

Constrained DET (Diffusive Equilibration in Thin films) probes equipped with 75 sampling layers of agarose gel (DGT Research(©)) were used to sample bottom and pore waters in marine sediment with a 2 mm vertical resolution. After retrieval, each piece of hydrogel, corresponding to 25 µL, was introduced into 1 mL of colorimetric reagent (CR) solution consisting of formic acid and bromophenol blue. After the elution/reaction time, absorbance of the latter mixture was read at 590 nm and compared to a calibration curve obtained with the same protocol applied to mini DET probes soaked in sodium hydrogen carbonate standard solutions. This method allows rapid alkalinity determinations for the small volumes of anoxic pore water entrapped into the gel. The method was assessed on organic-rich coastal marine sediments from Thau lagoon (France). Alkalinity values in the overlying waters were in agreement with data obtained by classical sampling techniques. Pore water data showed a progressive increase of alkalinity in the sediment from 2 to 10 mmol kg(-1), corresponding to anaerobic respiration in organic-rich sediments. Moreover, replicates of high-resolution DET profiles showed important lateral heterogeneity at a decimeter scale. This underlines the importance of high-resolution spatial methods for alkalinity profiling in coastal marine systems.

Laboratory screening for electrolyte abnormalities and anemia in bulimia nervosa: a controlled study.

OBJECTIVE: Abnormal eating patterns and recurrent purging behaviors can result in significant medical complications. The purpose of this study was to assess the frequency of abnormalities in clinical laboratory tests in patients with bulimia nervosa who reported being otherwise in good health. METHODS: Subjects included nonhospitalized women (N = 74) who met criteria for bulimia nervosa outlined in the 3rd Rev. ed. of the Diagnostic and Statistical Manual of Mental Disorders. They also reported use of self-induced vomiting and/or laxatives as compensatory behaviors (purging subtype). The control group (N = 110) included female volunteers with no history of a psychiatric disorder. All subjects reported being in good medical health, were medication free, and were in a normal weight range. Blood samples were analyzed in the hospital clinical laboratory. RESULTS: Compared with controls, patients showed more frequent occurrence of low values for serum potassium (6.8% vs. 0.9%; p <.05) and chloride (8.1% vs. 0.9%; p <.02). Electrolyte abnormalities occurred most often in patients with frequent bulimic episodes. Study groups did not differ significantly in frequency of abnormal hemoglobin concentrations. DISCUSSION: These results help to clarify the expected frequency of electrolyte abnormalities in individuals with bulimia nervosa who report otherwise good medical health. The substantial frequency of hypokalemia and hypochloremia underscores the importance of an appropriate medical assessment for individuals with this disorder.

Characterization of the adrenoleukodystrophy-related (ALDR, ABCD2) gene promoter: inductibility by retinoic acid and forskolin.

The adrenoleukodystrophy-related gene (ALDR, ABCD2) is a candidate modifier gene and a potential therapeutic target for X-linked adrenoleukodystrophy (ALD), a severe neurodegenerative disease. The ALDR gene is the closest homologue of the ALD gene, which encodes a peroxisomal ABC transporter involved in the catabolism of very-long-chain fatty acids. Administration of fenofibrate upregulates ALDR expression in rodent liver. As a step toward understanding ALDR transcriptional regulation, the mouse and human 5' regions were characterized. The human and mouse genes share a 500-bp conserved region that contains potential Sp1- and AP-2-binding sites but no TATA box. Analysis of the 5'-flanking region of ALDR using a luciferase reporter system revealed that 1.3 kb of human or mouse 5'-upstream region has functional promoter activity. In these transfection experiments, promoter activity of both human and mouse genes could be upregulated by 9-cis-retinoic acid and forskolin, while no effect of PPARalpha could be detected.

FHL2, a novel tissue-specific coactivator of the androgen receptor.

The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors. However, the exact mechanisms by which nuclear receptor-cofactor interactions result in tissue-specific gene regulation are unclear. Here we characterize a novel tissue-specific coactivator for the androgen receptor (AR), which is identical to a previously reported protein FHL2/DRAL with unknown function. In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus. FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor. In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2. Taken together, our data demonstrate that FHL2 is the first LIM-only coactivator of the AR with a unique tissue-specific expression pattern.

Serotonin function following remission from bulimia nervosa.

Abnormal serotonergic regulation in bulimia nervosa is thought to contribute to recurrent binge eating, depressed mood, and impulsivity. To follow-up on previous studies showing decreased neuroendocrine responses in symptomatic patients, this study assessed serotonin-mediated prolactin responses in individuals who had remitted from bulimia nervosa. Subjects included 21 women with a history of bulimia nervosa and 21 healthy female controls, as well as an additional comparison group of 19 women with current bulimia nervosa. Placebo-controlled neuroendocrine response studies utilized a single oral dose (60 mg) of the indirect serotonin agonist d,l-fenfluramine. For the bulimia nervosa remitted group, the fenfluramine-stimulated elevation in serum prolactin concentration was not significantly different from the response in healthy controls, but was significantly larger than the response in patients with current bulimia nervosa (p < .01). These findings suggest that diminished serotonergic neuroendocrine responsiveness in bulimia nervosa reflects a state-related abnormality. The results are discussed in relationship to recent reports indicating that some alterations in central nervous system serotonin regulation may persist in symptomatically recovered individuals.

Decreased serum leptin in bulimia nervosa.

The eating disorder bulimia nervosa has been associated with impaired satiety, decreased resting metabolic rate, and abnormal neuroendocrine regulation. Preclinical studies suggest that such alterations could be associated with impaired leptin function. Thus, the goal of this study was to assess whether leptin function is decreased in bulimia nervosa. Serum leptin levels measured in women with bulimia nervosa (n = 18) and in women who had maintained stable recovery from bulimia nervosa (n = 15) were compared with values in healthy female controls (n = 20). Subjects were studied during the follicular phase of their menstrual cycle after an overnight fast and bed rest. Baseline serum samples were analyzed for leptin concentration by RIA. Subject groups were matched for age and body weight. Analysis of covariance, adjusting for percent body fat, demonstrated abnormally low serum leptin levels in the bulimia nervosa group (P: = 0.02), with a trend toward an inverse correlation between frequency of binge episodes and serum leptin concentration (P: < 0.1). Additionally, the remitted patient group demonstrated abnormally low leptin values (P: = 0.01). These results are consistent with the hypothesis that decreased leptin function may be associated with alterations in eating patterns, metabolic rate, and neuroendocrine regulation in bulimia nervosa.

Salivary gland enlargement and elevated serum amylase in bulimia nervosa.

BACKGROUND: Clinical reports have described salivary gland enlargement in bulimia nervosa, particularly in patients with elevated serum amylase concentration. The goal of the current study was to provide a controlled comparison of salivary gland size in patients with bulimia nervosa and healthy volunteers. METHODS: Subjects included 17 women with bulimia nervosa and 21 healthy female control subjects. Dimensions of the parotid and submandibular salivary glands were estimated by ultrasonography. Blood samples for amylase measurement were obtained after overnight fast. RESULTS: Parotid gland size was enlarged 36% in patients with bulimia nervosa in comparison to control subjects (p < .01). For the patient group, salivary gland size was significantly correlated with frequency of bulimic symptoms and with serum amylase concentration. CONCLUSIONS: These results provide new quantitative data demonstrating increased salivary gland size in bulimia nervosa. Further studies are needed to evaluate factors responsible for salivary gland enlargement and hyperamylasemia in this disorder.

Role of nitric oxide in pancreatic tumour growth: in vivo and in vitro studies.

Nitric oxide (NO), an endogenous free radical, has been implicated in a wide range of biological functions. NO is generated enzymatically from the terminal guanidinonitrogen of L-arginine by nitric oxide synthase (NOS). Despite intensive investigations, the role of NO--either as the primary product of the L-arginine/NOS pathway or provided from the NO donor sodium nitroprusside (SNP)--in carcinogenesis and tumour cell growth remains unclear and controversial. The objective of this study was to examine the growth effects of NO on a ductal pancreatic adenocarcinoma in the rat and on a human pancreatic tumour cell line (HA-hpc2). In vivo, both SNP and endogenous induction of NO by endotoxins [lipopolysaccharide (LPS)] plus L-arginine significantly reduced the tumour growth. To investigate the mechanisms of NO anti-tumour growth action, the effects of either the SNP or L-arginine/NOS pathway were analysed on the HA-hpc2 cell line. Nitrite/nitrate production, NOS activity and iNOS expression [assessed by reverse transcription-polymerase chain reaction (RT-PCR)] were tested and related to growth (assessed by [3H]thymidine incorporation assay) and apoptosis (assessed by internucleosomal DNA cleavage). SNP exerted a dual effect on tumour cells: stimulation of the proliferation up to 1 mM and inhibition at higher concentrations. These effects were related to NO production. Both proliferative and cytostatic responses were inhibited by NO scavenger 2-phenyl-4,4,5,5-tetramethyl-hemidazoline-1-oxyl3-oxide (carboxy-PTIO). The marked apoptotic DNA fragmentation induced by SNP was also abolished by PTIO association. Unlike macrophages, the human pancreatic tumour cells did not seem to express intrinsically the L-arginine/NOS pathway. Macrophages were activated by HA-hpc2 cells as well as by LPS plus cytokines [interleukin (IL)-1beta plus tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma]. In HA-hpc2/macrophage co-cultures, NOS activity and inducible NOS (iNOS) transcription were stimulated, whereas an antiproliferative response was observed. These effects were related to both macrophage amount and NO production. Addition of LPS plus cytokines to co-cultures doubled iNOS activity, nitrite/nitrate production and tumoricidal effect. These data suggest the involvement of NO in pancreatic tumour growth and support the fact that generation of high levels of NO with potential production of endogenous reactive nitrogen intermediates may contribute to induction of apoptosis and tumour growth inhibition.

Mirror expression of adrenoleukodystrophy and adrenoleukodystrophy related genes in mouse tissues and human cell lines.

The adrenoleukodystrophy and adrenoleukodystrophy related proteins belong to a new family of half ATP-binding cassette transporters which are localized within the peroxisomal membrane and whose functions are still unknown. They could possibly homo- or heterodimerize resulting in transporters with similar or distinct functions. The expression of adrenoleukodystrophy and adrenoleukodystrophy related genes was studied at the mRNA and protein levels in adult mouse tissues and several human cell lines. We found that adrenoleukodystrophy and adrenoleukodystrophy related genes have strikingly different expression in most mouse tissues and human cell lines analyzed, indicating that adrenoleukodystrophy and adrenoleukodystrophy related proteins do not function as obligatory partners but might rather fulfill similar metabolic functions in different tissues.

Decreased serotonin function in bulimia nervosa.

BACKGROUND: Evidence that serotonin-active antidepressant medications decrease binge eating in patients with bulimia nervosa has fueled interest in the hypothesis that abnormal serotonergic neurotransmitter function contributes to symptoms of the disorder. To evaluate this hypothesis, we employed pharmacological challenge testing to compare serotonin function in patients with bulimia nervosa and healthy controls. METHODS: Neuroendocrine response patterns were compared for 15 nonhospitalized, medication-free, normal-weight women with bulimia nervosa and 14 age-matched healthy female controls. Behavioral assessment included ratings of eating disorder symptoms, depression, and anxiety. Serotonergic response patterns were assessed by measuring the increase in serum prolactin concentration during 5 hours following single-dose, oral administration of 60 mg of d,l-fenfluramine hydrochloride (Pondimin). RESULTS: For patients with bulimia nervosa, the fenfluramine-stimulated increase in serum prolactin concentration was significantly less than for controls. Within the patient group, the frequency of binge eating episodes during the 4 weeks prior to the study exhibited a significant inverse correlation with serotonin-stimulated prolactin secretion. CONCLUSION: Our study provides new evidence that impaired central nervous system serotonergic responsiveness may contribute to the onset or maintenance of abnormal eating patterns in patients with bulimia nervosa.

The effects of dieting on plasma tryptophan concentration and food intake in healthy women.

Although many people diet, relatively few dieters are successful in maintaining weight loss. The extent to which dieting behavior might dampen satiety responses normally mediated by the neurotransmitter serotonin remains uncertain. This study tested the hypothesis that dieting behavior decreases the availability of plasma tryptophan (TRP) and the ratio of TRP to other branched-chain amino acids (BCAA) that compete for entry into the central nervous system (CNS). This effect could diminish the CNS concentration of TRP, the amino acid precursor for serotonin synthesis, thus interfering with serotonin-mediated influences on food intake. Using a fixed-order design, 15 healthy, normal-weight women were studied longitudinally during an ad lib dietary intake phase and subsequent reduced-calorie diet phase. Physiological and behavioral measures were collected at baseline, at the end of the ad lib-intake phase, and at the end of the 4-week study diet phase. Food intake was measured by a single-item test meal. Plasma TRP and TRP: sigma BCAA significantly decreased following the study diet compared to baseline (p < 0.05). Change in TRP and TRP: sigma BCAA significantly correlated with decrease in body weight (p < 0.01). No significant relationship was observed between postdiet change in TRP or TRP: sigma BCAA ratio and postdiet change in test meal food intake, with covariation for weight loss. The observed decreases in plasma TRP and TRP: sigma BCAA extend previous reports suggesting that dieting behavior may diminish central serotonin function through a reduction in precursor availability.

Research update on serotonin function in bulimia nervosa and anorexia nervosa.

The extent to which dysregulation of serotonin function in the central nervous system may contribute to core symptoms in patients with bulimia nervosa and anorexia nervosa is currently an area of intensive psychobiological investigation. Preclinical and clinical studies have demonstrated the involvement of the neurotransmitter serotonin in the regulation of food intake, suggesting that impaired serotonin-mediated satiety signals could contribute to patterns of recurrent binge eating. Other symptom patterns in patients with eating disorders, including mood dysregulation, impulsivity, and obsessionality, as well as therapeutic response to serotonergic agents, suggest involvement of serotonergic pathways. With a primary focus on serotonin function, this article reviews clinical studies of neuroendocrine and behavioral response to pharmacological challenges, levels of neurotransmitter metabolite in cerebrospinal fluid, and platelet studies. Controlled clinical trials involving pharmacological treatment with serotonergic medications are summarized. Considerations for future research are discussed.

Medications in the treatment of eating disorders.

Effective planning for medication treatment in patients with bulimia nervosa and anorexia nervosa is based on a comprehensive clinical assessment, including a careful review of comorbid psychiatric disorders and response to treatments for previous episodes of the disorder. Although most patients with bulimia nervosa are offered a trial of psychotherapy, significant results of controlled trials have contributed to an increased role for medications in the treatment of patients with this disorder. Pharmacologic treatment of anorexia nervosa has similarities to that of treatment-resistant depression, with the clinician turning to open trials and clinical reports for clues to rational management. As described in this article, considerations of potential side effects and medical complications are likely to play an important role in guiding the choice of medication used for treatment of patients with eating disorders.

A modified therapeutic community for the dually diagnosed. Greenhouse Program at Bellevue Hospital.

Techniques used in therapeutic communities may be applicable to patients dually diagnosed with mental illness and a psychoactive substance use disorder (PSUD). This study was designed to evaluate the demographics, course, and outcome for 100 patients treated in one such residential program. One hundred indigent male patients admitted to a drug-free therapeutic community for the dually diagnosed were studied on admission and over the course of their treatment, and subjects were monitored throughout their stays on the basis of observed urine toxicology tests and a clinical assessment of drug or alcohol use. The mean age of the patients was 33.8 years, and the average length of stay was 121.0 days. Thirty-three of the patients completed the full 6-month program and moved on to another stable living environment. Only 12 patients had urine toxicologies positive for illicit drugs or alcohol while in the program. These findings support the possibility of applying the residential drug-free therapeutic community to dually diagnosed patients.