Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.     

Conservation significantly improves wetland conditions: evaluation of playa wetlands in different conservation status

This study assessed the conditions of wetland hydrology, hydrophyte and soil under different state and federal conservation programs, and then identified the restorable potential of conserved playas. The distribution of hydrology and hydrophyte were geospatially examined through annual tracking the quantity and quality of wetlands on historical hydric soil footprints under different conservation programs in the Rainwater Basin in Nebraska, USA during 2004–2015. The results show that the historical hydric soil footprints with the conservation programs had significantly better performance in ponded water and hydrophyte than non-conserved wetlands. The yearly average of ponded water areas within footprints varies at 12.59% for the Waterfowl Production Areas (WPAs), 14.78% for Wildlife Management Areas (WMAs), 27.37% for Wetlands Reserve Program’s conservation easements (WRPs), and 1.86% for non-conserved wetlands, respectively. The yearly average of hydrophyte plant community coverage within footprints reaches at 77.51% for WPAs, 79.28% for WMAs, and 66.53% for WRPs, and 8.82% for non-conserved hydric footprints. Within conserved lands, Massie/Water soil series demonstrated the prominent ability to hold ponding water, especially in the ponded footprints with higher ponding frequency. Nevertheless, the proportion of Fillmore, Rusco or Butler soil series roughly decreased when the ponding water frequency increased. The areas, with high likelihood to be restored, are the places between annual ponding/hydrophyte covered areas and 11 years’ maximized ponding/hydrophyte areas.

     

Skin surface cooling improves orthostatic tolerance following prolonged head-down bed rest

Prolonged exposure to microgravity, as well as its ground-based analog, head-down bed rest (HDBR), reduces orthostatic tolerance in humans. While skin surface cooling improves orthostatic tolerance, it remains unknown whether this could be an effective countermeasure to preserve orthostatic tolerance following HDBR. We therefore tested the hypothesis that skin surface cooling improves orthostatic tolerance after prolonged HDBR. Eight subjects (six men and two women) participated in the investigation. Orthostatic tolerance was determined using a progressive lower-body negative pressure (LBNP) tolerance test before HDBR during normothermic conditions and on day 16 or day 18 of 6° HDBR during normothermic and skin surface cooling conditions (randomized order post-HDBR). The thermal conditions were achieved by perfusing water (normothermia ～34°C and skin surface cooling ～12-15°C) through a tube-lined suit worn by each subject. Tolerance tests were performed after ～30 min of the respective thermal stimulus. A cumulative stress index (CSI; mmHg LBNP·min) was determined for each LBNP protocol by summing the product of the applied negative pressure and the duration of LBNP at each stage. HDBR reduced normothermic orthostatic tolerance as indexed by a reduction in the CSI from 1,037 ± 96 mmHg·min to 574 ± 63 mmHg·min (P < 0.05). After HDBR, skin surface cooling increased orthostatic tolerance (797 ± 77 mmHg·min) compared with normothermia (P < 0.05). While the reduction in orthostatic tolerance following prolonged HDBR was not completely reversed by acute skin surface cooling, the identified improvements may serve as an important and effective countermeasure for individuals exposed to microgravity, as well as immobilized and bed-stricken individuals.     

Effects of DOM photochemistry on bacterial metabolism and CO2 evasion during falling water in a humic and a whitewater river in the Brazilian Amazon

In the Amazon river system, the source of the large quantity of CO₂ evading from river surfaces remains unidentified. Photochemical transformation of dissolved organic matter (DOM) into dissolved inorganic carbon (DIC) and low molecular weight organic acids (LMWOAs) is a promising candidate. Few studies in the Amazon river system, and river systems in general, have attempted to quantify the contribution of these specific photoproducts to CO₂ evasion. We conducted photochemical degradation and ¹⁴C addition experiments to measure the rate of production and the impact on bacterial metabolism, respectively, in the black water Rio Negro and in the white water Rio Solimões during low water. We found statistically significant production of both photoproducts in the Rio Negro and none in Rio Solimões. We also found that two photochemically produced LMWOAs??acetic and formic acid??may play a significant role in bacterial metabolism in both rivers. Based on our experimental results, we estimate that photochemically produced CO₂, acetic acid and formic acid alone contribute to only 0.5% of the CO₂ evading from the Rio Negro. Due to our experimental set-up, analytical methods and time of sampling, we caution that our estimate is very conservative. More extensive research is needed before drawing conclusions on the contribution of photochemistry to CO₂ evasion from river surfaces of the Amazon basin.     

Large ring polymers align FtsZ polymers for normal septum formation

Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria. Mutation of sepF results in deformed division septa. It is unclear how SepF contributes to the synthesis of normal septa. We have studied SepF by electron microscopy (EM) and found that the protein assembles into very large (～50 nm diameter) rings. These rings were able to bundle FtsZ protofilaments into strikingly long and regular tubular structures reminiscent of eukaryotic microtubules. SepF mutants that disturb interaction with FtsZ or that impair ring formation are no longer able to align FtsZ filaments in vitro, and fail to support normal cell division in vivo. We propose that SepF rings are required for the regular arrangement of FtsZ filaments. Absence of this ordered state could explain the grossly distorted septal morphologies seen in sepF mutants.     

MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.     

N-methyl-D-aspartate receptor subunit NR3a expression and function in principal cells of the collecting duct

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ～80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.     

The conservation value of restored landfill sites in the East Midlands, UK for supporting bird communities

There has been a rapid decline of grassland bird species in the UK over the last four decades. In order to stem declines in biodiversity such as this, mitigation in the form of newly created habitat and restoration of degraded habitats is advocated in the UK biodiversity action plan. One potential restored habitat that could support a number of bird species is re-created grassland on restored landfill sites. However, this potential largely remains unexplored. In this study, birds were counted using point sampling on nine restored landfill sites in the East Midlands region of the UK during 2007 and 2008. The effects of restoration were investigated by examining bird species composition, richness, and abundance in relation to habitat and landscape structure on the landfill sites in comparison to paired reference sites of existing wildlife value. Twelve bird species were found in total and species richness and abundance on restored landfill sites was found to be higher than that of reference sites. Restored landfill sites support both common grassland bird species and also UK Red List bird species such as skylark Alauda arvensis, grey partridge Perdix perdix, lapwing Vanellus vanellus, tree sparrow, Passer montanus, and starling Sturnus vulgaris. Size of the site, percentage of bare soil and amount of adjacent hedgerow were found to be the most influential habitat quality factors for the distribution of most bird species. Presence of open habitat and crop land in the surrounding landscape were also found to have an effect on bird species composition. Management of restored landfill sites should be targeted towards UK Red List bird species since such sites could potentially play a significant role in biodiversity action planning.     

Aquaculture fouling: Efficacy of potassium monopersulphonate triple salt based disinfectant (Virkon® Aquatic) against Ciona intestinalis

With the increasing spread of invasive marine species and their detrimental effects on aquaculture operations globally, mitigation strategies need to be optimized to mitigate economic impacts. The efficacy of a potassium monopersulphonate triple salt based disinfectant used in the aquaculture industry (Virkon? Aquatic at 0.5-5%) was evaluated against the solitary tunicate Ciona intestinalis, as well as the susceptibility of three different age groups of C. intestinalis to the treatment and the effect of the disinfectant on mussel mortality. Younger C. intestinalis were most affected by all treatments, and almost all immersion applications significantly decreased the biomass of C. intestinalis compared to untreated plates. Disinfectant solutions of ≥ 1% reduced biomass below pre-treatment levels. Mussel mortality was low, especially for solutions <3%. C. intestinalis should be treated 4 weeks post-settlement to maximize antifouling treatment effects. Immersion in 3% disinfectant for 30 s reduced the biomass of C. intestinalis by up to 89% and would be feasible in field applications using existing treatment equipment.