Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

Tyrosine nitration moderates the peptidase activity of human methionyl aminopeptidase 2

Methionyl aminopeptidase 2 (MetAP2) plays an important role in the regulation of angiogenesis. This study examined whether nitration of MetAP2 alters its enzymatic activity in vitro. The activity of unmodified, nitrated and oxidised MetAP2 was assessed and it was found that nitration significantly reduced its ability to cleave a chromogenic substrate. Mass spectrometry analysis identified Tyr336 as a nitrated residue in MetAP2. Structural and evolutionary analysis indicate that this is an important residue for MetAP2 activity. Combined, the results show that the activity of MetAP2 is reduced by nitration and raise the possibility that nitration of MetAP2 is a mechanism contributing to endothelial dysfunction.     

Discovery of liver-targeted inhibitors of stearoyl-CoA desaturase (SCD1)

Inhibitors based on a benzo-fused spirocyclic oxazepine scaffold were discovered for stearoyl-coenzyme A (CoA) desaturase 1 (SCD1) and subsequently optimized to potent compounds with favorable pharmacokinetic profiles and in vivo efficacy in reducing the desaturation index in a mouse model. Initial optimization revealed potency preferences for the oxazepine core and benzylic positions, while substituents on the piperidine portions were more tolerant and allowed for tuning of potency and PK properties. After preparation and testing of a range of functional groups on the piperidine nitrogen, three classes of analogs were identified with single digit nanomolar potency: glycine amides, heterocycle-linked amides, and thiazoles. Responding to concerns about target localization and potential mechanism-based side effects, an initial effort was also made to improve liver concentration in an available rat PK model. An advanced compound 17m with a 5-carboxy-2-thiazole substructure appended to the spirocyclic piperidine scaffold was developed which satisfied the in vitro and in vivo requirements for more detailed studies.     

Bicyclic and tricyclic heterocycle derivatives as histamine H3 receptor antagonists for the treatment of obesity

A novel series of non-imidazole bicyclic and tricyclic histamine H₃ receptor antagonists has been discovered. Compound 17 was identified as a centrally penetrant molecule with high receptor occupancy which demonstrates robust oral activity in rodent models of obesity. In addition compound 17 possesses clean CYP and hERG profiles and shows no behavioral changes in the Irwin test.     

Fine‐scale temporal adaptation within a salmonid population: mechanism and consequences

We demonstrate a clear example of local adaptation of seasonal timing of spawning and embryo development. The consequence is a population of pink salmon that is segmented into spawning groups that use the same limited habitat. We synthesize published observations with results of new analyses to demonstrate that genetic variation of these traits results in survival differentials related to that variation, and that density‐dependent embryo mortality and seasonally variable juvenile mortality are a mechanism of selection. Most examples of local adaptation in natural systems depend on observed correlations between environments and fitness traits, but do not fully demonstrate local adaptation: that the trait is genetically determined, exhibits different fitness in common environments or across different environments, and its variation is mechanistically connected to fitness differences. The geographic or temporal scales of local adaptation often remain obscure. Here, we show that heritable, fine‐scale differences of timing of reproductive migration in a pink salmon (Oncorhynchus gorbuscha) resulted in temporal structure that persisted several generations; the differences enable a density‐dependent population to pack more spawners into limited spawning habitat, that is, enhance its fitness. A balanced trade‐off of survivals results because embryos from early‐migrating fish have a lower freshwater survival (harsh early physical conditions and disturbance by late spawners), but emigrant fry from late‐migrating fish have lower marine survivals (timing of their vernal emergence into the estuarine environment). Such fine‐scale local adaptations increase the genetic portfolio of the populations and may provide a buffer against the impacts of climate change.     

High dietary salt decreases antioxidant defenses in the liver of fructose-fed insulin-resistant rats

In this study we investigated the hypothesis that a high-salt diet to hyperinsulinemic rats might impair antioxidant defense owing to its involvement in the activation of sodium reabsorption to lead to higher oxidative stress. Rats were fed a standard (CON), a high-salt (HS), or a high-fructose (HF) diet for 10 weeks after which, 50% of the animals belonging to the HF group were switched to a regimen of high-fructose and high-salt diet (HFS) for 10 more weeks, while the other groups were fed with their respective diets. Animals were then euthanized and their blood and liver were examined. Fasting plasma glucose was found to be significantly higher (approximately 50%) in fructose-fed rats than in the control and HS rats, whereas fat liver also differed in these animals, producing steatosis. Feeding fructose-fed rats with the high-salt diet triggered hyperinsulinemia and lowered insulin sensitivity, which led to increased levels of serum sodium compared to the HS group. This resulted in membrane perturbation, which in the presence of steatosis potentially enhanced hepatic lipid peroxidation, thereby decreasing the level of antioxidant defenses, as shown by GSH/GSSG ratio (HFS rats, 7.098±2.1 versus CON rats, 13.2±6.1) and superoxide dismutase (HFS rats, 2.1±0.05 versus CON rats, 2.3±0.1%), and catalase (HFS rats, 526.6±88.6 versus CON rats, 745.8±228.7 U/mg ptn) activities. Our results indicate that consumption of a salt-rich diet by insulin-resistant rats may lead to regulation of sodium reabsorption, worsening hepatic lipid peroxidation associated with impaired antioxidant defenses.     

Effects of social disruption in elephants persist decades after culling

Background

Multi-level fission-fusion societies, characteristic of a number of large brained mammal species including some primates, cetaceans and elephants, are among the most complex and cognitively demanding animal social systems. Many free-ranging populations of these highly social mammals already face severe human disturbance, which is set to accelerate with projected anthropogenic environmental change. Despite this, our understanding of how such disruption affects core aspects of social functioning is still very limited.

Results

We now use novel playback experiments to assess decision-making abilities integral to operating successfully within complex societies, and provide the first systematic evidence that fundamental social skills may be significantly impaired by anthropogenic disruption. African elephants (Loxodonta africana) that had experienced separation from family members and translocation during culling operations decades previously performed poorly on systematic tests of their social knowledge, failing to distinguish between callers on the basis of social familiarity. Moreover, elephants from the disrupted population showed no evidence of discriminating between callers when age-related cues simulated individuals on an increasing scale of social dominance, in sharp contrast to the undisturbed population where this core social ability was well developed.

Conclusions

Key decision-making abilities that are fundamental to living in complex societies could be significantly altered in the long-term through exposure to severely disruptive events (e.g. culling and translocation). There is an assumption that wildlife responds to increasing pressure from human societies only in terms of demography, however our study demonstrates that the effects may be considerably more pervasive. These findings highlight the potential long-term negative consequences of acute social disruption in cognitively advanced species that live in close-knit kin-based societies, and alter our perspective on the health and functioning of populations that have been subjected to anthropogenic disturbance.

     

Noisy Galvanic Vestibular Stimulation Modulates the Amplitude of EEG Synchrony Patterns

Noisy galvanic vestibular stimulation has been associated with numerous cognitive and behavioural effects, such as enhancement of visual memory in healthy individuals, improvement of visual deficits in stroke patients, as well as possibly improvement of motor function in Parkinson’s disease; yet, the mechanism of action is unclear. Since Parkinson’s and other neuropsychiatric diseases are characterized by maladaptive dynamics of brain rhythms, we investigated whether noisy galvanic vestibular stimulation was associated with measurable changes in EEG oscillatory rhythms within theta (4–7.5 Hz), low alpha (8–10 Hz), high alpha (10.5–12 Hz), beta (13–30 Hz) and gamma (31–50 Hz) bands. We recorded the EEG while simultaneously delivering noisy bilateral, bipolar stimulation at varying intensities of imperceptible currents – at 10, 26, 42, 58, 74 and 90% of sensory threshold – to ten neurologically healthy subjects. Using standard spectral analysis, we investigated the transient aftereffects of noisy stimulation on rhythms. Subsequently, using robust artifact rejection techniques and the Least Absolute Shrinkage Selection Operator regression and cross-validation, we assessed the combinations of channels and power spectral features within each EEG frequency band that were linearly related with stimulus intensity. We show that noisy galvanic vestibular stimulation predominantly leads to a mild suppression of gamma power in lateral regions immediately after stimulation, followed by delayed increase in beta and gamma power in frontal regions approximately 20–25 s after stimulation ceased. Ongoing changes in the power of each oscillatory band throughout frontal, central/parietal, occipital and bilateral electrodes predicted the intensity of galvanic vestibular stimulation in a stimulus-dependent manner, demonstrating linear effects of stimulation on brain rhythms. We propose that modulation of neural oscillations is a potential mechanism for the previously-described cognitive and motor effects of vestibular stimulation, and noisy galvanic vestibular stimulation may provide an additional non-invasive means for neuromodulation of functional brain networks.