Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

Mapping genetic modifiers of survival in a mouse model of Dravet syndrome

Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage‐gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage‐gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss‐of‐function mutations in SCN1A result in Dravet syndrome, a severe infant‐onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a^+/?) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a^+/? mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a^+/? mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a^+/? mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a^+/? mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA‐seq analysis of strain‐dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a^+/? mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.     

Estimation of genetic variability and heritability for biofuel feedstock yield in several populations of switchgrass

Information on heritability and predicted gains from selection for increased biomass yield for ethanol production in switchgrass is limited and may vary among breeding populations. The purpose of this study was to estimate heritability and predicted gains from selection for higher biomass yield within a lowland ecotype switchgrass population, Southern Lowland 93 (SL‐93), and two upland ecotype switchgrass populations, Southern Upland Northern Upland Early Maturing (SNU‐EM) and Southern Upland Northern Upland Late Maturing (SNU‐LM). Narrow‐sense heritabilities (h_n²) for biomass yield in each of the three populations were estimated via progeny–parent regression analysis. Half‐sib (HS) progeny families from 130 randomly selected plants from the SL‐93 population were evaluated for biomass yield in replicated trials in 2002 and 2003. Clonal parent plants were evaluated for biomass yield in separate environments to provide unbiased h_n² estimates from progeny–parent regression. Yield differences were highly significant among SL‐93 HS progenies within and over years. For the SL‐93 population, h_n² estimates were 0.13 and 0.12 based on individual plant and phenotypic family mean (PFM) selection, respectively. Predicted genetic gains (ΔG) per selection cycle were 0.15 kg dry matter (dm) plant^?1 and 0.10 kg dm plant^?1 for PFM and individual plant selection methods, respectively. For the SNU‐EM and SNU‐LM populations, year and year × HS family effects were highly significant (P < 0.01) and the HS family effect over years was nonsignificant (P < 0.05). However, HS family effects were highly significant within respective years (P < 0.01). Estimates of h_n² for the SNU‐EM and SNU‐LM populations based on PFM and individual plant selection were similar, ranging from 0.44 to 0.47; ΔG per selection cycle ranged from 0.22 to 0.33 kg dm plant^?1. The magnitudes of the estimates of additive genetic variation suggest that selection for higher biomass yield should be possible. The substantial effect of environment on biomass yields in the upland populations and the failure of families to respond similarly over years stress the importance of adequately testing biomass yield over years to assess yield.     

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h² = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol .     

Involvement of A2A receptors in anxiolytic,locomotor and motivational properties of ethanol in mice

We have shown previously that mice lacking the adenosine A_2A receptor (A_2AR) generated on a CD1 background self‐administer more ethanol and exhibit hyposensitivity to acute ethanol. We aimed to investigate if the increased propensity of A_2A^?/? mice to consume ethanol is associated with an altered sensitivity in the motivational properties of ethanol in the conditioned place preference (CPP) and conditioned taste aversion (CTA) paradigms and with an altered development of sensitization to the locomotor effects of ethanol. We also tested their sensitivity to the anxiolytic effects of ethanol. Our results show that A_2A^?/? mice produced on a CD1 background displayed a reduced ethanol‐induced CPP and an increased sensitivity to the anxiolytic and locomotor‐stimulant effects of ethanol, but they did not show alteration in ethanol‐induced CTA and locomotor sensitization. Ethanol‐induced CPP, ethanol consumption and the locomotor effects of ethanol were also tested in A_2A^?/? mice produced on a C57BL/6J background. Our results emphasized the importance of the genetic background because alteration in ethanol consumption and preference, ethanol‐induced CPP and locomotor‐stimulant effects were not found in knockout mice produced on the alcohol‐preferring C57BL/6J genetic background. Finally, the A_2AR agonist, 2‐p‐(2‐carboxyethyl)‐phenylethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680), reduced ethanol consumption and preference in C57BL/6J mice. In conclusion, A_2AR deficiency in mice generated on a CD1 background leads to high ethanol consumption that is associated with an increased sensitivity to the locomotor‐stimulant/anxiolytic effects of ethanol and a decrease in ethanol‐induced CPP.     

Functional analysis of chick heparan sulfate 6‐O‐sulfotransferases in limb bud development

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O‐sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O‐sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS‐6‐O‐sulfotransferases (HS6ST‐1 and HS6ST‐2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf‐8 and Fgf‐10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST‐2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf‐8 and Fgf‐10 expressions than that achieved with HS6ST‐1 RNAi. HS6ST‐1 RNAi and HS6ST‐2 RNAi caused a significant but distinct reduction in the levels of different 6‐O‐sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6‐O‐sulfation of HS play essential roles in chick limb bud development.     

Distinct ethanol drinking microstructures in two replicate lines of mice selected for drinking to intoxication

The High Drinking in the Dark (HDID) mice have been selectively bred for reaching high blood ethanol concentrations (BECs) following the limited access Drinking in the Dark (DID) test. We have shown previously that mice from the first HDID replicate line (HDID‐1) drink in larger, but not longer, ethanol drinking bouts than the low‐drinking HS/Npt control mice when consuming modest amounts in the DID test. Here, we assessed drinking microstructure in HDID‐1 mice during binge‐like levels of ethanol intake using a lickometer system. Mice from both HDID replicates (HDID‐1 and ‐2) and HS mice were also given three DID tests (single‐bottle ethanol, two‐bottle choice and single‐bottle saccharin) using a continuously recording BioDAQ system to determine whether there are selection‐dependent changes in drinking microstructure. Larger ethanol bout size in the HDID‐1 mice than the HS mice was found to be due to a larger lick volume in these mice. HDID‐1 and HDID‐2 mice were also seen to have different drinking microstructures that both resulted in high intake and high BECs. The HDID‐1 mice drank in larger ethanol bouts than HS, whereas HDID‐2 mice drank in more frequent bouts. This pattern was also seen in two‐bottle choice DID. The HDID‐2 mice had a high bout frequency for all fluid types tested, whereas the large bout size phenotype of the HDID‐1 mice was specific to alcohol. These findings suggest that selection for drinking to intoxication has resulted in two distinct drinking microstructures, both of which lead to high BECs and high ethanol intake.     

Detecting selection signatures between Duroc and Duroc synthetic pig populations using high‐density SNP chip

The development of high throughput genotyping techniques has facilitated the identification of selection signatures of pigs. The detection of genomic selection signals in a population subjected to differential selection pressures may provide insights into the genes associated with economically and biologically important traits. To identify genomic regions under selection, we genotyped 488 Duroc (D) pigs and 155 D × Korean native pigs (DKNPs) using the Porcine SNP70K BeadChip. By applying the F_ST and extended haplotype homozygosity (EHH‐Rsb) methods, we detected genes under directional selection associated with growth/stature (DOCK7, PLCB4, HS2ST1, FBP2 and TG), carcass and meat quality (TG, COL14A1, FBXO5, NR3C1, SNX7, ARHGAP26 and DPYD), number of teats (LOC100153159 and LRRC1), pigmentation (MME) and ear morphology (SOX5), which are all mostly near or at fixation. These results could be a basis for investigating the underlying mutations associated with observed phenotypic variation. Validation using genome‐wide association analysis would also facilitate the inclusion of some of these markers in genetic evaluation programs.     

High‐Sensitivity C‐Reactive Protein in Japanese Patients with Type 2 Diabetes

Objective: To assess the relationship between high‐sensitivity (HS) C‐reactive protein (CRP) and metabolic syndrome (MetS) or atherosclerosis and to assess effects of strict metabolic control on the degree of inflammation and MetS in patients with type 2 diabetes. Research Methods and Procedures: Four hundred thirteen patients with diabetes were enrolled in the cross‐sectional study. Of these 413 patients, 161 patients were further admitted for 2.4 ± 0.4 weeks (mean ± SD) to investigate the change in HS‐CRP or other parameters under strict metabolic control. Results: Log‐transformed HS‐CRP value (log HS‐CRP) was strongly correlated with BMI (r = 0.448, p < 0.01). Log HS‐CRP was also correlated with the presence of MetS or each component of MetS. Furthermore, a positive significant trend in HS‐CRP levels was shown with an increasing number of MetS components (p < 0.05). Log HS‐CRP showed a significant positive correlation with carotid artery intima‐media thickness (IMT) (r = 0.152, p < 0.01). In multiple step‐wise regression analysis, BMI, hemoglobin A_1c, right IMT, duration of diabetes, and triglyceride were selected as explanatory variables for log HS‐CRP (R² = 0.412). Under strict metabolic control, HS‐CRP was significantly (p < 0.01) lower, together with lower levels of other markers for MetS. The change in HS‐CRP was significantly correlated with the change in BMI (r = 0.161, p = 0.04). Discussion: In subjects with type 2 diabetes, HS‐CRP levels are related to MetS and subclinical atherosclerosis. Strict weight management and metabolic control were associated with a reduction in HS‐CRP levels, and changes in HS‐CRP were related to changes in weight, supporting the hypothesis that lifestyle modification reduces inflammation and the risk of CHD.     

Enantioselectivity in Developmental Toxicity of rac‐metalaxyl and R‐metalaxyl in Zebrafish (Danio rerio) Embryo

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac‐metalaxyl and R‐metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac‐metalaxyl and R‐metalaxyl, and the LC₅₀s at 96 h are 416.41 (353.91, 499.29) mg · L^‐1 and 320.650 (279.80, 363.46) mg · L^‐1, respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic–pituitary–gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic–pituitary–thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac‐metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R‐metalaxyl. And likewise, rac‐metalaxyl only caused the upregulation of dio2, while R‐metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489–494, 2016. © 2016 Wiley Periodicals, Inc.     

Iridoids from Pedicularis verticillata and Their Anti‐Complementary Activity

Three new iridoids named as pediverticilatasin A – C ( 1 – 3 , resp.), together with five known iridoids ( 4 – 8 , resp.) were isolated from the whole plants of Pedicularis verticillata. The structures of three new compounds were identified as (1S,7R)‐1‐ethoxy‐1,5,6,7‐tetrahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4(3H)‐one ( 1 ), (1S,4aS,7R,7aS)‐1‐ethoxy‐1,4a,5,6,7,7a‐hexahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4‐carboxylic acid ( 2 ), (1S,4aS,7R,7aS)‐1‐ethoxy‐1,4a,5,6,7,7a‐hexahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4‐carbaldehyde ( 3 ). Their structures were elucidated on the basis of spectroscopic methods and compared with the NMR spectra data in the literature. All compounds were evaluated for their anti‐complementary activity on the classical pathway of the complement system in vitro. Among which, compounds 1 , 3 , and 6 exhibited anti‐complementary effects with CH₅₀ values ranging from 0.43 to 1.72 mm , which are plausible candidates for developing potent anti‐complementary agents.     

A mouse model for visualization of GABAB receptors

GABA_B receptors are the G‐protein‐coupled receptors for the neurotransmitter γ‐aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA_B1a and GABA_B1b, which combine with GABA_B2 subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA_B1 gene to generate transgenic mice expressing GABA_B1a and GABA_B1b subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA_B1‐eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA_B1 proteins in the brain and in peripheral tissue. Crossing the GABA_B1‐eGFP BAC transgene into the GABA_B1^?/? background restores pre and postsynaptic GABA_B functions, showing that the GABA_B1‐eGFP fusion proteins substitute for the lack of endogenous GABA_B1 proteins. Finally, we demonstrate that the GABA_B1‐eGFP fusion proteins replicate the temporal expression patterns of native GABA_B receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA_B receptors. genesis 47:595–602, 2009. © 2009 Wiley‐Liss, Inc.     

Continuous multistep versus fed‐batch production of ethanol and xylitol in a simulated medium of sugarcane bagasse hydrolyzates

Co‐cultures for simultaneous production of ethanol and xylitol were studied under different operation bioreactor modes using Candida tropicalis IEC5‐ITV and Saccharomyces cerevisiae ITV01‐RD in a simulated medium of sugarcane bagasse hydrolyzates. Xylitol and ethanol tolerance by S. cerevisiae and C. tropicalis, respectively, was evaluated. The results showed that C. tropicalis was sensitive to ethanol concentrations up to 30 g/L, while xylitol had no effect on S. cerevisiae viability and metabolism. The best condition found for simultaneous culture was S. cerevisiae co‐culture and C. tropicalis sequential cultivation at 24 h. Under these conditions, productivity and yield for ethanol were Q_EtOH = 0.72 g L^?1 h^?1 and Y_EtOH/s = 0.37 g/g, and for xylitol, Q_XylOH = 0.10 g L^?1 h^?1 and Y_XylOH/S = 0.31 g/g, respectively; using fed‐batch culture, the results were Q_EtOH = 0.87 g L^?1 h^?1 and Y_EtOH/s = 0.44 g L^?1 h^?1, and Q_EtOH = 0.27 g L^?1 h^?1 and Y_EtOH/s = 0.57 g/g, respectively. Maximum volumetric productivity in continuous multistep cultures of ethanol and xylitol was at dilution rates of 0.131 and 0.074 h^?1, respectively. Continuous multistep production, Q_EtOH increased up to 50% more than in fed‐batch culture, even though xylitol yield remained unchanged.     

Identification of potentially critical genes in the development of heart failure after ST-segment elevation myocardial infarction (STEMI)

Heart failure (HF) remains a common complication after acute ST-segment elevation myocardial infarction (STEMI). Here, we aim to identify critical genes related to the developed HF in patients with STEMI using bioinformatics analysis. The microarray data of GSE59867, including peripheral blood samples from nine patients with post-infarct HF and eight patients without post-infarct HF, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between HF and non-HF groups were screened by LIMMA package. Functional enrichment analyses of DEGs were conducted, followed by construction of a protein-protein interaction (PPI) network. The dynamic messenger RNA (mRNA) level of the hub genes during the follow-up was analyzed to further elucidate their role in HF development. A total of 58 upregulated and 75 downregulated DEGs were screen out. They were mainly enriched in biological processes about inflammatory response, extracellular matrix organization, response to cAMP, immune response, and positive regulation of cytosolic calcium ion concentration. Pathway analysis revealed that the DEGs were also involved in hematopoietic cell lineage, pathways in cancer, and extracellular matrix-receptor interaction. In the PPI network consisting of 58 nodes and 72 interactions, CXCL8 (degree = 15), THBS1 (degree = 8), FOS (degree = 7), and ITGA2B (degree = 6) were identified as the hub genes. In the comparison of patients with and without post-infarct HF, the mRNA level of these hub genes were all higher within 30 days but reached similar at 6 months after STEMI. In conclusion, CXCL8, THBS1, FOS, and ITGA2B may play important roles in the development of HF after acute STEMI.     

Antisense regulation by transposon‐derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon‐derived paralogous RNAs, RNA‐257_1–4, show extended complementarity to the 3′ UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA‐257₁ and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA‐257₁. Insertion of the 1183 3′ UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.     

Effects of elevated atmospheric [CO2] on instantaneous transpiration efficiency at leaf and canopy scales in Eucalyptus saligna

Rising atmospheric concentrations of CO₂ (C_a) can reduce stomatal conductance and transpiration rate in trees, but the magnitude of this effect varies considerably among experiments. The theory of optimal stomatal behaviour predicts that the ratio of photosynthesis to transpiration (instantaneous transpiration efficiency, ITE) should increase in proportion to C_a. We hypothesized that plants regulate stomatal conductance optimally in response to rising C_a. We tested this hypothesis with data from young Eucalyptus saligna Sm. trees grown in 12 climate‐controlled whole‐tree chambers for 2 years at ambient and elevated C_a. Elevated C_a was ambient + 240 ppm, 60% higher than ambient C_a. Leaf‐scale gas exchange was measured throughout the second year of the study and leaf‐scale ITE increased by 60% under elevated C_a, as predicted. Values of leaf‐scale ITE depended strongly on vapour pressure deficit (D) in both CO₂ treatments. Whole‐canopy CO₂ and H₂O fluxes were also monitored continuously for each chamber throughout the second year. There were small differences in D between C_a treatments, which had important effects on values of canopy‐scale ITE. However, when C_a treatments were compared at the same D, canopy‐scale ITE was consistently increased by 60%, again as predicted. Importantly, leaf and canopy‐scale ITE were not significantly different, indicating that ITE was not scale‐dependent. Observed changes in transpiration rate could be explained on the basis that ITE increased in proportion to C_a. The effect of elevated C_a on photosynthesis increased with rising D. At high D, C_a had a large effect on photosynthesis and a small effect on transpiration rate. At low D, in contrast, there was a small effect of C_a on photosynthesis, but a much larger effect on transpiration rate. If shown to be a general response, the proportionality of ITE with C_a will allow us to predict the effects of C_a on transpiration rate.