Working with cattle slurry on farms: emission and dispersion of hydrogen sulfide gas during stirring.

Over the past 15 years, there have been numerous fatalities related to working with animal slurry. Working with cattle slurry releases toxic gases, in particular, hydrogen sulphide (H2S), which can cause acute central nervous system toxicity, breathing difficulties, and death if exposed to high concentrations. Real-time measurements of H2S gas were taken over distance and time, during the stirring of cattle slurry on farms. Gas was measured at eight slurry stores with differing typical configurations of indoor or outdoor stores and with or without slatted flooring. Highest H2S gas levels were measured from indoor stores under slatted floors, and generally at positions closest to the stirrer or the point of maximum stirring, with levels decreasing with distance from source. Most of the data indicate H2S gas levels increase very rapidly after stirring starts, and mostly decline to baseline levels within 30 min post start of stirring. There were, however, circumstances where gas levels remained high and only started to decline once the stirrer had stopped. H2S gas levels at all farms, at all positions measured were consistently below 10 ppm within 30 min of the stirrer being stopped. The current data highlight areas of the farm and ways of working that have the potential for workers and others to be at risk of exposure to toxic slurry gases. The area should be left to ventilate naturally for at least 30 min after the stirrer has been stopped before re-entering buildings. Influencing the design of stirring equipment and future slurry stores would likely reduce the risk of worker exposure to slurry gases.

Dietary fat and corticosterone levels are contributing factors to meal anticipation.

Daily restricted access to food leads to the development of food anticipatory activity and metabolism, which depends upon an as yet unidentified food-entrainable oscillator(s). A premeal anticipatory peak in circulating hormones, including corticosterone is also elicited by daily restricted feeding. High-fat feeding is associated with elevated levels of corticosterone with disrupted circadian rhythms and a failure to develop robust meal anticipation. It is not clear whether the disrupted corticosterone rhythm, resulting from high-fat feeding contributes to attenuated meal anticipation in high-fat fed rats. Our aim was to better characterize meal anticipation in rats fed a low- or high-fat diet, and to better understand the role of corticosterone in this process. To this end, we utilized behavioral observations, hypothalamic c-Fos expression, and indirect calorimetry to assess meal entrainment. We also used the glucocorticoid receptor antagonist, RU486, to dissect out the role of corticosterone in meal anticipation in rats given daily access to a meal with different fat content. Restricted access to a low-fat diet led to robust meal anticipation, as well as entrainment of hypothalamic c-Fos expression, metabolism, and circulating corticosterone. These measures were significantly attenuated in response to a high-fat diet, and animals on this diet exhibited a postanticipatory rise in corticosterone. Interestingly, antagonism of glucocorticoid activity using RU486 attenuated meal anticipation in low-fat fed rats, but promoted meal anticipation in high-fat-fed rats. These findings suggest an important role for corticosterone in the regulation of meal anticipation in a manner dependent upon dietary fat content.

Free-Wilson and structural approaches to co-optimizing human and rodent isoform potency for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been a target of intensive research efforts across the pharmaceutical industry, due to its potential for the treatment of type II diabetes and other elements of the metabolic syndrome. To demonstrate the value of 11ß-HSD1 in preclinical models, we required inhibitors with good potency against both human and rodent isoforms. Herein, we describe our efforts to understand how to co-optimize human and murine potency within the (5-hydroxy-2-adamantyl)-pyrimidine-5-carboxamide series. Two approaches are described-a data-driven (Free-Wilson) analysis and a structure-based design approach. The conclusions from these approaches were used to inform an efficient campaign to design compounds with consistently good human/murine potency within a logD(7.4) range of 1-3. Compounds 20 and 26 demonstrated good rodent PK, which allowed us to demonstrate a PK/PD relationship in rat and mouse. We then evaluated 26 against glycemic and body weight end points in murine disease models, where it demonstrated glucose and body weight efficacy at 300 mg/kg/day but only body weight efficacy at 50 mg/kg/day, despite providing >90% target engagement in the liver.

Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (AGRP)83-132: interaction between AGRP83-132 and melanocortin receptors cannot be influenced by syndecan-3.

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg(79)-Glu(80)-Pro(81)-Arg(82). The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP(83-132) is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP(25-47) and AGRP(50-80) had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.