Epigenetics of amphetamine-induced sensitization: HDAC5 expression and microRNA in neural remodeling

Background: Histone deacetylase (HDAC) activities modify chromatin structure and play a role in learning and memory during developmental processes. Studies of adult mice suggest HDACs are involved in neural network remodeling in brain repair, but its function in drug addiction is less understood. We aimed to examine in vivo HDAC5 expression in a preclinical model of amphetamine-induced sensitization (AIS) of behavior. We generated specific contrast agents to measure HDAC5 levels by in vivo molecular contrast-enhanced (MCE) magnetic resonance imaging (MRI) in amphetamine-naïve mice as well as in mice with AIS. To validate the MRI results we used ex vivo methods including in situ hybridization, RT-PCR, immunohistochemistry, and transmision electron microscopy. Methods: We compared the expression of HDAC5 mRNA in an acute exposure paradigm (in which animals experienced a single drug exposure [A1]) and in a chronic-abstinence-challenge paradigm (in which animals were exposed to the drug once every other day for seven doses, then underwent 2 weeks of abstinence followed by a challenge dose [A7WA]). Control groups for each of these exposure paradigms were given saline. To delineate how HDAC5 expression was related to AIS, we compared the expression of HDAC5 mRNA at sequences where no known microRNA (miR) binds (hdac5AS2) and at sequences where miR-2861 is known to bind (miD2861). We synthesized and labeled phosphorothioated oligonucleic acids (sODN) of hdac5AS2 or miD2861 linked to superparamagentic iron oxide nanoparticles (SPION), and generated HDAC5-specific contrast agents (30 ± 20 nm, diameter) for MCE MRI; the same sequences were used for primers for TaqMan® analysis (RT-qPCR) in ex vivo validation. In addition, we used subtraction R2* maps to identify regional HDAC5 expression. Results: Naïve C57black6 mice that experience acute exposure to amphetamine (4 mg/kg, by injection intraperitoneally) show expression of both total and phosphorylated (S259) HDAC5 antigens in GFAP+ and GFAP− cells, but the appearance of these cells was attenuated in the chronic paradigm. We found that MCE MRI reports HDAC5 mRNA with precision in physiological conditions because the HDAC5 mRNA copy number reported by TaqMan analysis was positively correlated (with a linear coefficient of 1.0) to the ΔR2* values (the frequency of signal reduction above background, 1/s) measured by MRI. We observed SPION-mid2861 as electron dense nanoparticles (EDNs) of less than 30 nm in the nucleus of the neurons, macrophages, and microglia, but not in glia and endothelia. We found no preferential distribution in any particular type of neural cells, but observed scattered EDNs of 60–150 nm (dia) in lysosomes. In the acute paradigm, mice pretreated with miD2861 (1.2 mmol/kg, i.p./icv) exhibited AIS similar to that exibited by mice in the chronic exposure group, which exhibited null response to mid2861 pretreatment. Moreover, SPION-miD2861 identified enhanced HDAC5 expression in the lateral septum and the striatum after amphetamine, where we found neurprogenitor cells coexpressing NeuN and GFAP. Conclusions: We conclude that miD2681 targets HDAC5 mRNA with precision similar to that of RT-PCR. Our MCE MRI detects RNA-bound nanoparticles (NPs) in vivo, and ex vivo validation methods confirm that EDNs do not accumulate in any particular cell type. As HDAC5 expression may help nullify AIS and identify progenitor cells, the precise delivery of miD2861 may serve as a vehicle for monitoring network remodeling with target specificity and signal sensitivity after drug exposure that identifies brain repair processes in adult animals. Electronic supplementary material The online version of this article (doi:10.1186/s12929-016-0294-8) contains supplementary material, which is available to authorized users

A Censored Bayesian Hierarchical Model For Precipitation

Modelling of precipitation, including extremes, is important for hydrological and agricultural applications. Traditionally, because of large sample properties for data over a large threshold value, generalised Pareto (GP) distributions are often used for modelling extreme rainfall. It can be shown that under certain conditions the generalised hyperbolic (GH) distributions can approximate the power law decay of the GP distribution in the tails. Given their flexible form, this raises the possibility that distributions from the GH family serve as a model for the entire rainfall distribution thus avoiding the need to select a threshold. In this paper, we use a flexible censored hierarchical model that leverages the GH distribution to accommodate data subject to heavy tails and an excessive number of zeros. The fitted model allows estimation of probabilities and return periods of the rainfall extremes, and it produces narrower credible intervals in the tails than the traditional GP method. The model not only fits the tails of the rainfall distribution, but fits the whole distribution very well. It also efficiently represents short-term dependencies in the data so it is suitable for evaluating duration over and below thresholds as well as duration of zero rainfall.Comment: Under review at Environmentric

Selection for Chloroquine-Sensitive Plasmodium Falciparum by Wild Anopheles Arabiensis in Southern Zambia

Background: The emergence of parasite drug resistance, especially Plasmodium falciparum, persists as a major obstacle for malaria control and elimination. To develop effective public health containment strategies, a clear understanding of factors that govern the emergence and spread of resistant parasites in the field is important. The current study documents selection for chloroquine-sensitive malaria parasites by wild Anopheles arabiensis in southern Zambia. Methods. In a 2,000-sq km region, mosquitoes were collected from human sleeping rooms using pyrethrum spray catches during the 2006 malaria transmission season. After morphological examination and molecular confirmation, vector mosquitoes were dissected to separate head and thorax from the abdominal section, followed by PCR screening for P. falciparum infection. Human residents of all ages were tested for P. falciparum parasitaemia by microscopy and PCR. Plasmodium falciparum infections were genotyped at the chloroquine resistance-conferring amino acid codon 76 of the PfCRT gene, using PCR and restriction enzyme digestion. Results: In the human population there was nearly 90% prevalence of the chloroquine-resistant PfCRT K76T mutant, with no significant differences in polymorphism among smear-positive and smear-negative (submicroscopic) infections (p = 0.323, n = 128). However, infections in both abdominal and salivary gland phases of the An. arabiensis vector exhibited wild type K76-bearing parasites with up to 9X higher odds (OR (95% CI): 9 (3.7-20.2), p \u3c 0.0005, n = 125), despite having been acquired from humans within a few weeks. Conclusions: Anopheles arabiensis selects for wild-type K76-bearing P. falciparum during both abdominal and salivary gland phases of parasite development. The rapid vectorial selection, also recently seen with antifolate resistance, is evidence for parasite fitness cost in the mosquito, and may underpin regional heterogeneity in the emergence, spread and waning of drug resistance. Understanding the nature and direction of vector selection could be instrumental for rational curtailment of the spread of drug resistance in integrated malaria control and elimination programmes

Parameter identifiability and model selection for partial differential equation models of cell invasion

When employing a mechanistic model to study biological systems, practical parameter identifiability is important for making predictions in a wide range of scenarios, as well as for understanding the mechanisms driving the system behaviour. We argue that parameter identifiability should be considered alongside goodness-of-fit and model complexity as criteria for model selection. To demonstrate, we use a profile likelihood approach to investigate parameter identifiability for four extensions of the Fisher--KPP model, given experimental data from a cell invasion assay. We show that more complicated models tend to be less identifiable, with parameter estimates being more sensitive to subtle differences in experimental procedures, and require more data to be practically identifiable. The results from identifiability analysis can inform model selection, as well as data collection and experimental design.Comment: 23 pages in main text, 21 pages in supplementary material

Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney.

BackgroundExcess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout.MethodssUA levels, fractional excretion of uric acid (FEUA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity.ResultsAfter 6 hours, a single 200-mg dose of lesinurad elevated FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting.ConclusionThe pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout

GABA-modulating bacteria of the human gut microbiota.

The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression

IKKβ is Essential for Adipocyte Survival and Adaptive Adipose Remodeling in Obesity

IκB kinase β (IKKβ), a central coordinator of inflammatory responses through activation of nuclear factor-κB (NF-κB), has been implicated as a critical molecular link between inflammation and metabolic disorders; however, the role of adipocyte IKKβ in obesity and related metabolic disorders remains elusive. Here we report an essential role of IKKβ in the regulation of adipose remodeling and adipocyte survival in diet-induced obesity. Targeted deletion of IKKβ in adipocytes does not affect body weight, food intake, and energy expenditure but results in an exaggerated diabetic phenotype when challenged with a high-fat diet (HFD). IKKβ-deficient mice have multiple histopathologies in visceral adipose tissue, including increased adipocyte death, amplified macrophage infiltration, and defective adaptive adipose remodeling. Deficiency of IKKβ also leads to increased adipose lipolysis, elevated plasma free fatty acid (FFA) levels, and impaired insulin signaling. Mechanistic studies demonstrated that IKKβ is a key adipocyte survival factor and that IKKβ protects murine and human adipocytes from HFD- or FFA-elicited cell death through NF-κB-dependent upregulation of antiapoptotic proteins and NF-κB-independent inactivation of proapoptotic BAD protein. Our findings establish IKKβ as critical for adipocyte survival and adaptive adipose remodeling in obesity

Super-Resolution Imaging of C-Type Lectin and Influenza Hemagglutinin Nanodomains on Plasma Membranes Using Blink Microscopy

AbstractDendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts