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

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions

Recurrent deletions of ULK4 in schizophrenia : a gene crucial for neuritogenesis and neuronal motility

Peer reviewedPublisher PD

Semen quality in relation to biomarkers of pesticide exposure.

We previously reported reduced sperm concentration and motility in fertile men in a U.S. agrarian area (Columbia, MO) relative to men from U.S. urban centers (Minneapolis, MN; Los Angeles, CA; New York, NY). In the present study we address the hypothesis that pesticides currently used in agriculture in the Midwest contributed to these differences in semen quality. We selected men in whom all semen parameters (concentration, percentage sperm with normal morphology, and percentage motile sperm) were low (cases) and men in whom all semen parameters were within normal limits (controls) within Missouri and Minnesota (sample sizes of 50 and 36, respectively) and measured metabolites of eight current-use pesticides in urine samples provided at the time of semen collection. All pesticide analyses were conducted blind with respect to center and case-control status. Pesticide metabolite levels were elevated in Missouri cases, compared with controls, for the herbicides alachlor and atrazine and for the insecticide diazinon [2-isopropoxy-4-methyl-pyrimidinol (IMPY)]; for Wilcoxon rank test, p = 0.0007, 0.012, and 0.0004 for alachlor, atrazine, and IMPY, respectively. Men from Missouri with high levels of alachlor or IMPY were significantly more likely to be cases than were men with low levels [odds ratios (ORs) = 30.0 and 16.7 for alachlor and IMPY, respectively], as were men with atrazine levels higher than the limit of detection (OR = 11.3). The herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and metolachlor were also associated with poor semen quality in some analyses, whereas acetochlor levels were lower in cases than in controls (p = 0.04). No significant associations were seen for any pesticides within Minnesota, where levels of agricultural pesticides were low, or for the insect repellent DEET (N,N-diethyl-m-toluamide) or the malathion metabolite malathion dicarboxylic acid. These associations between current-use pesticides and reduced semen quality suggest that agricultural chemicals may have contributed to the reduction in semen quality in fertile men from mid-Missouri we reported previously

X-ray, Optical, and Radio Observations of the Type II Supernovae 1999em and 1998S

Observations of the Type II-P (plateau) Supernova (SN) 1999em and Type IIn (narrow emission line) SN 1998S have enabled estimation of the profile of the SN ejecta, the structure of the circumstellar medium (CSM) established by the pre-SN stellar wind, and the nature of the shock interaction. SN 1999em is the first Type II-P detected at both X-ray and radio wavelengths. The Chandra X-ray data indicate non-radiative interaction of SN ejecta with a power-law density profile (rho \propto r^{-n} with n ~ 7) with a pre-SN wind with a low mass-loss rate of ~2 \times 10^{-6} M_sun/yr for a wind velocity of 10 km/sec, in agreement with radio mass-loss rate estimates. The Chandra data show an unexpected, temporary rise in the 0.4--2.0 keV X-ray flux at ~100 days after explosion. SN 1998S, at an age of >3 years, is still bright in X-rays and is increasing in flux density at cm radio wavelengths. Spectral fits to the Chandra data show that many heavy elements (Ne, Al, Si, S, Ar, and Fe) are overabundant with respect to solar values. We compare the observed elemental abundances and abundance ratios to theoretical calculations and find that our data are consistent with a progenitor mass of approximately 15-20 M_sun if the heavy element ejecta are radially mixed out to a high velocity. If the X-ray emission is from the reverse shock wave region, the supernova density profile must be moderately flat at a velocity ~10^4 km/sec, the shock front is non-radiative at the time of the observations, and the mass-loss rate is 1-2 \times 10^{-4} M_sun/yr for a pre-supernova wind velocity of 10 km/sec. This result is also supported by modeling of the radio emission which implies that SN 1998S is surrounded by a clumpy or filamentary CSM established by a high mass-loss rate, ~2 \times 10^{-4} M_sun/yr, from the pre-supernova star.Comment: 14 pages, 10 figures, accepted by ApJ, includes new data (one new obs. each of SN 1999em and SN 1998S), expanded discussion of spectral fit

Large-Scale Covariability Between Aerosol and Precipitation Over the 7-SEAS Region: Observations and Simulations

One of the seven scientific areas of interests of the 7-SEAS field campaign is to evaluate the impact of aerosol on cloud and precipitation (http://7-seas.gsfc.nasa.gov). However, large-scale covariability between aerosol, cloud and precipitation is complicated not only by ambient environment and a variety of aerosol effects, but also by effects from rain washout and climate factors. This study characterizes large-scale aerosol-cloud-precipitation covariability through synergy of long-term multi ]sensor satellite observations with model simulations over the 7-SEAS region [10S-30N, 95E-130E]. Results show that climate factors such as ENSO significantly modulate aerosol and precipitation over the region simultaneously. After removal of climate factor effects, aerosol and precipitation are significantly anti-correlated over the southern part of the region, where high aerosols loading is associated with overall reduced total precipitation with intensified rain rates and decreased rain frequency, decreased tropospheric latent heating, suppressed cloud top height and increased outgoing longwave radiation, enhanced clear-sky shortwave TOA flux but reduced all-sky shortwave TOA flux in deep convective regimes; but such covariability becomes less notable over the northern counterpart of the region where low ]level stratus are found. Using CO as a proxy of biomass burning aerosols to minimize the washout effect, large-scale covariability between CO and precipitation was also investigated and similar large-scale covariability observed. Model simulations with NCAR CAM5 were found to show similar effects to observations in the spatio-temporal patterns. Results from both observations and simulations are valuable for improving our understanding of this region's meteorological system and the roles of aerosol within it. Key words: aerosol; precipitation; large-scale covariability; aerosol effects; washout; climate factors; 7- SEAS; CO; CAM

Geographic differences in semen quality of fertile U.S. males.

Although geographic variation in semen quality has been reported, this is the first study in the United States to compare semen quality among study centers using standardized methods and strict quality control. We evaluated semen specimens from partners of 512 pregnant women recruited through prenatal clinics in four U.S. cities during 1999-2001; 91% of men provided two specimens. Sperm concentration, semen volume, and motility were determined at the centers, and morphology was assessed at a central laboratory. Study protocols were identical across centers, and quality control was rigorously maintained. Sperm concentration was significantly lower in Columbia, Missouri, than in New York, New York; Minneapolis, Minnesota; and Los Angeles, California. Mean counts were 58.7, 102.9, 98.6, and 80.8 X 10(6)/mL (medians 53.5, 88.5, 81.8, and 64.8 X 10(6)/mL) in Missouri, New York, Minnesota, and California, respectively. The total number of motile sperm was also lower in Missouri than in other centers: 113, 196, 201, and 162 X 10(6) in Missouri, New York, Minnesota, and California, respectively. Semen volume and the percent morphologically normal sperm did not differ appreciably among centers. These between-center differences remained significant in multivariate models that controlled for abstinence time, semen analysis time, age, race, smoking, history of sexually transmitted disease, and recent fever (all p-values < 0.01). Confounding factors and differences in study methods are unlikely to account for the lower semen quality seen in this mid-Missouri population. These data suggest that sperm concentration and motility may be reduced in semirural and agricultural areas relative to more urban and less agriculturally exposed areas

Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells

Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1), an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC). In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA). We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP) assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter

Deterministic evolution and stringent selection during preneoplasia

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours