Search

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.

Some of the neurobehavioral deficits identified in children with Fetal Alcohol Spectrum Disorders have been recapitulated in a binge model of gestational third trimester-equivalent ethanol exposure, in which Sprague-Dawley rats are intragastrically intubated between post-natal day (PD) 4 and PD9 with high doses of ethanol. In this model, the ameliorating effects of choline administration on hippocampal-dependent behaviors altered by ethanol have also been extensively documented. In the present study, we investigated the effects of ethanol (5g/kg/day) and/or choline (100mg/kg/day) on morphometric parameters of CA1 pyramidal neurons by Golgi-Cox staining followed by Neurolucida tracing and analysis. We found that ethanol increased apical dendrite complexity in male and female pups neonatally exposed to ethanol. Ethanol did not significantly affect basal dendrite parameters in female and male rats. Interestingly, choline treatments decreased basal dendrites’ length, number, and maximal terminal distance in male pups. When pups were co-treated with ethanol and choline, choline did not rescue the effect of ethanol. In conclusion, ethanol increases while choline decreases dendritic length and arborization of hippocampal CA1 neurons in PD9 rats. We hypothesize that developmental ethanol exposure induces a premature maturation of neurons, leading to early restriction of neuronal plasticity while choline treatments delay the normal program of neuronal maturation and therefore prolong the window of maximal plasticity. Choline does not prevent the effects of developmental alcohol exposure on hippocampal pyramidal neurons’ morphology characterized in the present study, although whether prolonged choline administration after developmental ethanol exposure rectifies ethanol damage remains to be assessed.

Anaerobic digestion is a long-established technology for the valorization of diverse organic wastes with concomitant generation of valuable resources. However, mono-digestion (i.e., anaerobic digestion using one feedstock) suffers from challenges associated with feedstock characteristics. Co-digestion using multiple feedstocks provides the potential to overcome these limitations. Significant research and development efforts have highlighted several inherent merits of co-digestion, including enhanced digestibility due to synergistic effects of co-substrates, better process stability, and higher nutrient value of the produced co-digestate. However, studies focused on the underlying effects of diverse co-feedstocks on digester performance and stability have not been synthesized so far. This review fills this gap by highlighting the limitations of mono-digestion and critically examining the benefits of co-digestion. Furthermore, this review discusses synergistic effect of co-substrates, characterization of microbial communities, the prediction of biogas production via different kinetic models, and highlights future research directions for the development of a sustainable biorefinery.

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

The efficacy of C4 grasses as feedstocks for liquid fuel production and their climate mitigation potential remain unresolved in the tropics. To identify highly convertible C4 grasses, we measured final fuels and postprocess biomass produced in two laboratory-scale conversion pathways across 12 species and varieties within the Poaceae (grass) family. Total mass, carbon, and energy in final fuels and postprocess biomass were assessed based on field mass and area-based production. Two lignocellulosic processes were investigated: (1) anaerobic digestion (AD) to methane and (2) hot water pretreatment and enzymatic hydrolysis (HWP-EH) to ethanol. We found AD converted lignocellulose to methane more efficiently in terms of carbon and energy compared to ethanol production using HWP-EH, although improvements to and the optimization of each process could change these contrasts. The resulting data provide design limitations for agricultural production and biorefinery systems that regulate these systems as net carbon sources or sinks to the atmosphere. Median carbon recovery in final fuels and postprocess biomass from the studied C4 grasses were ~5 Mg C ha−1 year−1 for both methane and ethanol, while median energy recovery was ~200 MJ ha−1 year−1 for ethanol and ~275 MJ ha−1 year−1 for methane. The highest carbon and energy recovery from lignocellulose was achieved during methane production from a sugarcane hybrid called energycane, with ~10 Mg C ha−1 year−1 and ~450 MJ ha−1 year−1 of carbon and energy recovered, respectively, from fuels and post-process biomass combined. Carbon and energy recovery during ethanol production was also highest for energycane, with ~9 Mg C ha−1 year−1 and ~350 MJ ha−1 year−1 of carbon and energy recovered in fuels and postprocess biomass combined. Although several process streams remain unresolved, agricultural production and conversion of C4 grasses must operate within these carbon and energy limitations for biofuel and bioenergy production to be an atmospheric carbon sink.

Global interest in renewable fuels is rapidly growing with particular emphasis on local energy growth and creating new energy feedstocks, specifically liquid fuel sources. However, the interactive effect of plant species/variety and growth environment on plant structural components, which may influence conversion efficiency and thus play an important role in optimizing the production of biofuels, is not fully understood. In this study cupric oxide (CuO) extractable lignin, which extracts and quantifies lignin-derived monomers, was determined for 25 cultivated and naturalized tropical perennial C4 grass varieties of napiergrass and Guinea grass that were under assessment for suitability as feedstocks for liquid fuel generation. Principal component analysis of CuO extractable lignin-derived monomers showed differences in composition between napiergrass and Guinea grass, as well as environmental differences within many, but not all napiergrass varieties. Among the samples tested, the greatest differences in lignin composition occurred specifically in vanillyl lignin structures. A wide range (1–2.5) in the ratio of cinnamyl to vanillyl structures (C/V), which often relates to enzymatic degradability in natural systems, was also found. It is expected that the observed CuO lignin differences representing the structure and bonding of lignin will relate to ease of chemical and enzymatic conversion and the effectiveness of pretreatment and conversion in biofuel application. We hypothesize that the C/V ratio of feedstock will positively relate to conversion efficiency and if supported, then compositional lignin metrics such as the C/V ratio could be a predictor to select for more easily degradable biomass for biofuel production.

This study examined the composition and anaerobic digestibility of the different plant parts of two high-yielding tropical energy crops, Energycane and Napier grass, collected across three locations and two seasons. Both biomass composition and biomethane yields varied significantly with crop types, plant parts and harvest seasons. In Energycane, specific methane yield (SMY) (Nm3 (kg VSadded)−1) was higher from stems (0.232 ± 0.003) than leaves (0.224 ± 0.003), while in Napier grass, SMY was higher from leaves (0.243 ± 0.002) than stems (0.168 ± 0.002). Energycane had higher specific and total (Nm3 ha−1 year−1) methane yields (0.230 ± 0.002 and 8749 ± 494, respectively) than Napier grass (0.192 ± 0.002 and 5575 ± 494, respectively). The SMYs from biomass correlated negatively with acid detergent fiber, cellulose and lignin content in the biomass. Energycane and Napier grass had lower specific but comparable total methane yields (TMYs) with maize. The ecological, economic and environmental merits associated with perennial crops suggest they could outperform maize as a substrate for bioenergy production.

Fetal alcohol spectrum disorders (FASD) encompasses all the consequences of maternal alcohol consumption during pregnancy in the offspring. Central nervous system dysfunctions and structural and functional brain abnormalities are the most deleterious consequences of prenatal alcohol exposure leading to cognitive and behavioral impairment that affect individuals throughout life. This chapter describes the cognitive, neurobehavioral, and brain structural abnormalities identified in humans following in utero alcohol exposure. This chapter also describes FASD animal models used in research to increase the understanding of the consequences of alcohol exposure during gestation on the developing brain. Finally, this chapter describes the cellular/molecular mechanisms involved in the developmental neurotoxicity of alcohol as well as the interventions that have been proposed and are currently under investigation to improve the consequences of in utero alcohol exposure.

Chronic alcohol abuse is associated with brain damage in a sex-specific fashion, but the mechanisms involved are poorly described and remain controversial. Previous results have suggested that astrocyte gene expression is influenced by ethanol intoxication and during abstinence in vivo. Here, bioinformatic analysis of astrocyte-enriched ethanol-regulated genes in vivo revealed ubiquitin pathways as an ethanol target, but with sexually dimorphic cytokine signaling and changes associated with brain aging in females and not males. Consistent with this result, astrocyte activation was observed after exposure in female but not male animals, with reduced S100β levels in the anterior cingulate cortex and increased GFAP(+) cells in the hippocampus. In primary culture, the direct effects of chronic ethanol exposure followed by recovery on sex-specific astrocyte function were examined. Male astrocyte responses were consistent with astrocyte deactivation with reduced GFAP expression during ethanol exposure. In contrast, female astrocytes exhibited increased expression of Tnf, reduced expression of the neuroprotective cytokine Tgfb1, disrupted bioenergetics and reduced excitatory amino acid uptake following exposure or recovery. These results indicate widespread astrocyte dysfunction in ethanol-exposed females and suggest a mechanism that may underlie increased vulnerability to ethanol-induced neurotoxicity in females.

Women are more sensitive to the harmful effects of alcohol (EtOH) abuse than men, yet the underlying mechanisms remain poorly understood. Previous gene expression analysis of the medial prefrontal cortex (mPFC) following a chronic intoxication paradigm using continuous 72 h vapor inhalation found that females, but not males, exhibit an inflammatory response at peak withdrawal that is associated with cell damage. Given that glucocorticoids can function as anti-inflammatories, are known to increase with EtOH exposure, and influence neurotoxicity, we hypothesized that males and females may exhibit an altered corticosterone (CORT) response following chronic intoxication. Analysis of serum CORT levels revealed the expected increase during withdrawal with no difference between males and females, while control males but not females exhibited higher CORT concentrations than naive animals. Glucocorticoid signaling characterized using focused qPCR arrays identified a sexually dimorphic response in the mPFC during withdrawal, particularly among astrocyte-enriched genes. These genes include aquaporin-1 (Aqp1), sphingosine kinase 1 (Sphk1) and connective tissue growth factor (Ctgf); genes associated with inflammatory signaling, and tissue damage and repair. Bioinformatic analysis also revealed activation of inflammatory signaling and cell death pathways in females. Confirmation studies showed that female mice exhibited significant neuronal degeneration within the anterior cingulate cortex (ACC). By contrast, EtOH exposure lead to a significant reduction in cell death in males. Thus, distinct glucocorticoid signaling pathways are associated with sexually dimorphic neurotoxicity, suggesting one mechanism by which EtOH-exposed females are particularly vulnerable to the damaging effects of alcohol in the CNS.

Energy is an indispensable part of modern society and can serve as one of the most important indicators of socio-economic development. Despite advancements in technology, however, some three billion people, primarily in the rural areas of developing countries, continue to meet their energy needs for cooking through traditional means by burning biomass resources (i.e., firewood, crop residues and animal dung) in crude traditional stoves. Such practices are known to be the source of significant environmental, social, economic and public health issues. To achieve sustainable development in these regions, it is imperative that access to clean and affordable (renewable) energy is made available. Within this context, upgrading existing biomass resources (i.e., animal manure, crop residues, kitchen waste and green wastes) to cleaner and more efficient energy carriers (such as biogas from anaerobic digestion) has unique potential to provide clean and reliable energy, while simultaneously preserving the local and global environment. In spite of its significant potential to serve developing nations, however, the high costs and lack of expertise in installation and maintenance of biogas technology preclude widespread adoption in geographically isolated communities. Concerted efforts from both governmental and non-governmental sectors are absolutely essential in facilitating modernization and dissemination of biogas technology to harness the inherent potential that is currently underutilized and unexploited. The intent of this paper seeks to highlight the present status, challenges, and potential of biogas technology to advocate for further research, development and dissemination of the concept in developing countries.

Background Binge ethanol (EtOH) intake during adolescence leads to an array of behavioral and cognitive consequences including elevated intake of EtOH during adulthood, with female mice showing greater susceptibility than males. Administration of the metabotropic glutamate receptor 5 (mGluR5) antagonist 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) has been shown to reduce EtOH self-administration in adult male mice, but little is known about its effect on female and adolescent mice. Methods MTEP (0, 10, 20 mg/kg, i.p.) was repeatedly administered to female and male, adult and adolescent C57BL/6J mice during binge sessions using the scheduled high alcohol consumption paradigm. Next, we assessed whether MTEP administration during binge altered the subsequent 24-hour EtOH intake following a period of abstinence. Finally, we investigated whether MTEP administration during binge followed by an abstinence period altered mRNA of glutamatergic genes within the nucleus accumbens of female mice. Results MTEP significantly decreased binge EtOH intake in all mice, but only female mice exhibited altered subsequent 24-hour EtOH intake. Interestingly, the alteration in subsequent EtOH intake in female animals was age dependent, with adolescent exposure to MTEP during binge decreasing 24-hour intake and adult exposure to MTEP during binge increasing 24-hour intake. Finally, while there were no effects of MTEP pretreatment on the genes examined, there was a robust age effect found during analysis of mGluR1 (Grm1), mGluR5 (Grm5), the NR2A subunit of the NMDA receptor (Grin2a), phosphatidylinositol 3-kinase (Pik3r1), mammalian target of rapamycin (Mtor), and extracellular signal-regulated kinase (Mapk1) mRNA, with adolescent female animals having lower expression than their adult counterparts. Conclusions Collectively, the present findings add to existing evidence implicating the contribution of long-term effects of adolescent binge drinking to enhance alcohol abuse in adulthood, while suggesting that mGluR5 antagonism may not be the best pharmacotherapy to treat binge alcohol consumption in female and adolescent animals.

Alcohol abuse is associated with neurological dysfunction, brain morphological deficits and frank neurotoxicity. Although these disruptions may be a secondary effect due to hepatic encephalopathy, no clear evidence of causality is available. This study examined whether a 72 h period of alcohol intoxication known to induce physical dependence, followed by a single withdrawal, was sufficient to induce signs of hepatic encephalopathy in male and female mice. Animals were continuously intoxicated via alcohol vapor inhalation, a procedure previously shown to induce significant neurotoxicity in female mice. At peak synchronized withdrawal (8 h following the end of alcohol exposure), blood samples were taken and levels of several liver-regulated markers and brain swelling were characterized. Glutathione levels were also determined in the medial frontal cortex (mFC) and hippocampus. Results revealed elevated levels of cholesterol, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and decreased levels of blood urea nitrogen and total bilirubin in alcohol-exposed male and female groups compared to controls. Brain water weight was not affected by alcohol exposure, though males tended to have slightly more water weight overall. Alcohol exposure led to reductions in tissue levels of glutathione in both the hippocampus and mFC which may indicate increased oxidative stress. Combined, these results suggest that hepatic encephalopathy does not appear to play a significant role in the neurotoxicity observed following alcohol exposure in this model.

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest.

Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.

Radial and longitudinal strain assessment in the carotid artery wall using speckle tracking

Improved prevention and treatment of drug addiction will require deeper understanding of genetic factors contributing to susceptibility to excessive drug use. Intravenous operant self-administration methods have greatly advanced understanding of behavioral traits related to addiction. However, these methods are not suitable for large-scale genetic experiments in mice. Selective breeding of mice can aggregate 'addiction alleles' in a model that has the potential to identify coordinated effects of multiple genes. We produced mouse lines that orally self-administer high (MAHDR) or low (MALDR) amounts of methamphetamine, representing the first demonstration of selective breeding for self-administration of any psychostimulant drug. Conditioned place preference and taste aversion results indicate that MAHDR mice are relatively more sensitive to the rewarding effects and less sensitive to the aversive effects of methamphetamine, compared to MALDR mice. These results validate the oral route of self-administration for investigation of the motivational effects of methamphetamine and provide a viable alternative to intravenous self-administration procedures. Gene expression results for a subset of genes relevant to addiction-related processes suggest differential regulation by methamphetamine of apoptosis and immune pathways in the nucleus accumbens of MAHDR and MALDR mice. In each line, methamphetamine reduced an allostatic state by bringing gene expression back toward 'normal' levels. Genes differentially expressed in the drug-naï ve state, including Slc6a4 (serotonin transporter), Htr3a (serotonin receptor 3A), Rela [nuclear factor kappaB (NFkappaB)] and Fos (cFos), represent candidates whose expression levels may predict methamphetamine consumption and susceptibility to methamphetamine reward and aversion.

While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways were more altered by alcohol. Examination of ethanol-regulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse.

It is well established that binge alcohol consumption produces alterations in Group 1 metabotropic glutamate receptors (mGlus) and related signaling cascades in the nucleus accumbens (NAC) of adult male mice, but female and adolescent mice have not been examined. Thus, the first set of studies determined whether repeated binge alcohol consumption produced similar alterations in protein and mRNA levels of Group 1 mGlu-associated signaling molecules in the NAC of male and female adult and adolescent mice. The adult (9 weeks) and adolescent (4 weeks) C57BL/6J mice were exposed to 7 binge alcohol sessions every 3rd day while controls drank water. Repeated binge alcohol consumption produced sexually divergent changes in protein levels and mRNA expression for Group 1 mGlus and downstream signaling molecules in the NAC, but there was no effect of age. Binge alcohol intake decreased mGlu5 levels in females, whereas it decreased indices of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), 4E-binding protein 1, and p70 ribosomal protein S6 kinase in males. Expression of genes encoding mGlu1, mGlu5, the NR2A subunit of the NMDA receptor, and Homer2 were all decreased by binge alcohol consumption in males, while females were relatively resistant (only phosphoinositide-dependent protein kinase 1 was decreased). The functional implication of these differences was investigated in a separate study by inhibiting mTOR in the NAC (via infusions of rapamycin) before binge drinking sessions. Rapamycin (50 and 100 ng/side) significantly decreased binge alcohol consumption in males, while consumption in females was unaffected. Altogether these results highlight that mTOR signaling in the NAC was necessary to maintain binge alcohol consumption only in male mice and that binge drinking recruits sexually divergent signaling cascades downstream of PI3K and presumably, Group 1 mGlus. Importantly, these findings emphasize that sex should be considered in the development of potential pharmacotherapeutic targets.

Terrestrial carbon (C) plays an important role in the global C cycle. Implementing agricultural practices that increase C fixation, into both above and below ground pools, will play an important role in mitigating global warming through atmospheric C sequestration. Though current agricultural practices tend to be optimized for production efficiency and not for environmental sustainability, the two need not be mutually exclusive. This study discusses the initial selection process of potential tropical C4 feedstocks and implementation of sustainable management practices at Hawaii Commercial & Sugar (HC&S) on Maui. Preliminary lignin analysis of 25 varieties of potential C4 grass feedstocks, through the use of the copper oxide (CuO) oxidation extraction method, has shown that lignin composition is variable between similar species and also varies based on environmental conditions. Lignin results in combination with total biomass yields were used as metrics for the evaluation and implementation of feedstocks in further trials. In three years under sustainable management practices, measured soil C stocks of napiergrass showed a substantial and sustained 29% increase in C compared to baseline. This C increase is expected to improve soil health, increase agricultural productivity, decrease water use, and create a net reduction in soil C emissions. Diversification into lignocellulosic biofuel feedstocks could further reduce C emissions by allowing HC&S to produce a C neutral fuel source for income generation and use on site. However, optimization of plant structural components (lignin, cellulose, and hemicellulose) and conversion efficiency will play an important role in the production of fuel from sustainably grown feedstocks. In addition, if lignin composition can be related to the ease of conversion into biofuels, then the CuO method may become an important tool for future feedstock selections in sustainably managed biorefinery systems.

This project had two main goals. The first goal was to evaluate several high yielding tropical perennial grasses as feedstock for biofuel production, and to characterize the feedstock for compatible biofuel production systems. The second goal was to assess the integration of renewable energy systems for Hawaii. The project focused on high-yield grasses (napiergrass, energycane, sweet sorghum, and sugarcane). Field plots were established to evaluate the effects of elevation (30, 300 and 900 meters above sea level) and irrigation (50%, 75% and 100% of sugarcane plantation practice) on energy crop yields and input. The test plots were extensive monitored including: hydrologic studies to measure crop water use and losses through seepage and evapotranspiration; changes in soil carbon stock; greenhouse gas flux (CO2, CH4, and N2O) from the soil surface; and root morphology, biomass, and turnover. Results showed significant effects of environment on crop yields. In general, crop yields decrease as the elevation increased, being more pronounced for sweet sorghum and energycane than napiergrass. Also energy crop yields were higher with increased irrigation levels, being most pronounced with energycane and less so with sweet sorghum. Daylight length greatly affected sweet sorghum growth and yields. One of the energy crops (napiergrass) wasmore » harvested at different ages (2, 4, 6, and 8 months) to assess the changes in feedstock characteristics with age and potential to generate co-products. Although there was greater potential for co-products from younger feedstock, the increased production was not sufficient to offset the additional cost of harvesting multiple times per year. The feedstocks were also characterized to assess their compatibility with biochemical and thermochemical conversion processes. The project objectives are being continued through additional support from the Office of Naval Research, and the Biomass Research and Development Initiative. Renewable energy assessments included: biomass feedstocks currently being produced by Hawaiian Commercial & Sugar Co., and possibilities of producing methane from agricultural and livestock wastes and the potential of photovoltaic systems for irrigation pumping at HC&S. Finally, the impact of a micro-hydroelectric system on a small-farm economics and the local community was assessed.« less

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 μM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

This article represents the proceedings of a symposium at the Research Society on Alcoholism meeting in Santa Barbara, California. The organizers/chairs were Kristine M. Wiren and Deborah A. Finn. Following a brief introduction by Deborah Finn, the presentations were (1) The Importance of Gender in Determining Expression Differences in Mouse Lines Selected for Chronic Ethanol Withdrawal Severity, by Kristine M. Wiren and Joel G. Hashimoto; (2) Sex Differences in Ethanol Withdrawal Involve GABAergic and Stress Systems, by Paul E. Alele and Leslie L. Devaud; (3) The Influence of Sex on Ethanol Consumption and Reward in C57BL/6 Mice, by Kimber L. Price and Lawrence D. Middaugh; and (4) Sex Differences in Alcohol Self-administration in Cynomolgus Monkeys, by Kathleen A. Grant.

This article summarizes the proceedings of a symposium presented at the 2004 annual meeting of the Research Society on Alcoholism in Vancouver, British Columbia, Canada. The organizers and chairs were William J. McBride and Michael F. Miles. The presentations were (1) Molecular Triangulation on Gene Expression Patterns in Behavioral Responses to Acute Ethanol, by Robnet T. Kerns; (2) Gene Expression in Limbic Regions After Ethanol Self-Infusion Into the Posterior Ventral Tegmental Area, by Zachary A. Rodd; (3) Microarray Analysis of CNS Limbic Regions of Inbred Alcohol-Preferring and -Nonpreferring rats and Effects of Alcohol Drinking, by Wendy N. Strother and Howard J. Edenberg; and (4) Microarray Analysis of Mouse Lines Selected for Chronic Ethanol Withdrawal Severity: The Convergence of Basal, Ethanol Regulated, and Proximity to Ethanol Quantitative Trait Loci to Identify Candidate Genes, by Joel G. Hashimoto and Kristine M. Wiren.

Background: Normative Doppler values and determinants for gender difference of right ventricular diastolic function in healthy subjects have not been fully elucidated. The purpose of this study is to verify the difference of right ventricular diastolic function between men and women at each age. Method: One hundred sixty nine male and 221 female in healthy subject, aged 20 to 90, were examined by both tricuspid inflow velocity and tricuspid annulus velocity by tissue Doppler imaging. All subjects were divided into 7 groups as 20s, 30s, 40s, 50s, 60s, 70s, 80s depend on their age. Results: The tricuspid E velocity was significantly decreased with age (p,0.0001). The tricuspid A velocity was significantly increased with age (p,0.0001). But both E and A velocity were no differences significantly in all generation between men and women (Table). Ratio of tricuspid E and A velocity (E/A) was significantly decreased with age. But no differences were observed in ratio of E/A in all generation between men and women. (20s: 2.0+0.6 vs 2.3+0.6, 30s: 1.9+0.5 vs 2.1+0.5, 40s: 1.6+0.4 vs 1.9+0.5, 50s: 1.3+0.3 vs 1.5+0.4, 60s: 1.2+0.2 vs 1.2+0.3, 70s: 1.1+0.3 vs 1.1+0.3, 80s: 1.2+0.3 vs 1.2+0.3, total: 1.4+0.5 vs 1.5+0.6). Early diastole velocity of tricuspid annulus (Ea) by tissue Doppler method was significantly decreased with age. But Ea was no differences significantly in all generation between men and women (Table). Conclusion: Our results provide the normative Doppler values of right ventricular diastolic function for age decades. There was no difference significantly of right ventricular diastolic function between men and women.

Ethanol abuse can lead to addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. There is evidence for contributions of both genotype and sex to alcoholism, but an understanding of the biological underpinnings is limited. Utilizing both sexes of genetic animal models with highly divergent alcohol withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) mice, the distinct contributions of genotype/phenotype and of sex during addiction stages on neuroadaptation were characterized. Transcriptional profiling was performed to identify expression changes as a consequence of chronic intoxication in the medial prefrontal cortex. Significant expression differences were identified on a single platform and tracked over a behaviorally relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more sensitive to ethanol with higher fold expression differences. Bioinformatics showed a strong effect of sex on the data structure of expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, differences were observed instead between the lines/phenotypes irrespective of sex. Confirmation of identified pathways showed distinct inflammatory signaling following intoxication at peak withdrawal, with a pro-inflammatory phenotype in females but overall suppression of immune signaling in males. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of stage- and sex-specific therapies for alcohol withdrawal and the maintenance of sobriety.

We report experimental results obtained by using a wide-gap type emulsion chamber flown in the first Japanese-Russo joint balloon project, called RUNJOB ( RU ssia- N ippon JO int B alloon-program). Two balloons were launched from Kamchatka in July 1995, and both were recovered successfully near the Volga River. The exposure time was 130 hours for the first flight and 168 hours for the second. The mean ceiling altitude, in both flights, was 32 km corresponding to 10 g/cm 2 . Total area of the emulsion chamber was 0.8 m 2 , and the thickness 0.385 and 2.28 collision m.f.p.'s for vertically incident proton- and iron-primaries, respectively. We detected 381 showers using Fuji-#200-type X-ray film; of these 174 showers were due to atmospheric secondary γ-rays, and the rest 207 came from nuclear components. The energy range covers 20∼200 TeV for proton-primary, 3∼30 TeV/nucleon for helium-primary, and 0.7∼5 TeV/nucleon for iron-primary. We give the energy spectra for various elements (proton, helium, …, iron) as well as the all-particle spectrum and the average mass of the cosmic-ray primaries.

The relative toxicity of the combined nonxylose components in prehydrolysates derived from three different lignocellulosic biomass feedstocks was determined. One woody (poplar) and two herbaceous (corn stover and switchgrass) feedstocks were dilute-acid pretreated under temperature and acid conditions chosen to optimize xylose recovery in the liquid prehydrolysate; xylose yields averaged 96,89,and 87% of theoretical for switchgrass,corn stover,and poplar,respectively. Prehydrolysates from each feedstock were neutralized,adjusted to equivalent xylose concentrations,and bioassayed for toxicity,using a standardized fermentation protocol withPichia stipitis NRRL 11545. Full time-courses for ethanol production (30-60 h) clearly illustrate the distinct inhibitory effects of prehydrolysates from different feedstocks. The relative toxicity of the prehydrolysates,ranked in order of decreasing toxicity,is poplar-derived prehydrolysates > switchgrass-derived prehydrolysates > corn stover-derived prehydrolysates. The inhibition of ethanol production appeared to be the result of a general slowdown of yeast metabolism,rather than the result of the production of alternative, nonethanol end products. Ethanol yields averaged 74,83,and 88% of control values for poplar,switchgrass,and corn stover prehydrolysates, respectively. Volumetric ethanol productivities (g ethanol L/h) averaged 32,70,and 102% of control values for poplar,switchgrass,and corn stover prehydrolysates,respectively. Ethanol productivities correlated closely with acetate concentrations in the prehydrolysates; however, regression lines correlating acetate concentrations and ethanol productivities were found to be feedstock-dependent.

Lignocellulosic biomass may be used as a potential renewable feedstock for biochemical production of ethanol as an alternative transportation fuel. However, cellulose, which is the major source of fermentable sugars in these materials, is protected by a network of lignin and hemicellulose. The dilute-sulfuric-acid pretreatment removes this protecting shield and makes the cellulose more susceptible to enzymatic digestion. In this study, three lignocellulosic feedstocks (i.e. corn stover, poplar and switchgrass) were pretreated with dilute sulfuric acid (0.6, 0.9 and 1.2% w/w) at relatively high temperatures (140, 160 and 180°C) in a Parr batch reactor. The hydrolysis of hemicellulose to its monomeric constituents and possible degradation of these monomers were modeled by a series of first-order reactions. The kinetic parameters of two mathematical models for predicting the percentage of xylan remaining in the substrate after pretreatment and the net xylose yield in the liquid stream were determined using the actual acid concentration in the reactor after accounting for the neutralization effect of the substrates.

Batch digestion experiments were conducted to determine the effects of inoculum:substrate ratio and initial medium pH on methane production from glucose. A ratio between the chemical oxygen demands (COD) of the substrate and inoculum solids (SISR) was used to quantify the food to microorganisms (F/M) ratio. Two periods of active methane production, separated by a period of inactivity, characterized the fermentation. The duration of inactivity was affected by both SISR and the initial medium pH. It decreased as SISR was decreased or when a higher initial pH was used. With a SISR of 4.8 and a pH of 7.2 or 7.0, the inactive period was eliminated, indicating satisfactory coupling of the acid- and methane-forming phases. Ultimate methane yield at a SISR of 4.8 was higher than at a SISR of 38.5.

Methane is the most abundant organic chemical in the earth's atmosphere, and its abundance is increasing with time and has reached levels not seen in recent geological history. Methane is produced both naturally and anthropogenically. One of teh sources of anthropogenic methane is manure from domesticated animals. A methodology has previously been developed to estimate the amount of methane generated from this source. This was done by estimating the methane conversion factor (MCF) typically achieved by various waste management systems. The present study was conducted to evaluate these MCF assumptions using dairy manure as the representative livestock manure. The MCFs for the most dominant of disposal methods, rangeland/pasture disposal, were much lower than the earlier estimates. Other waste management systems, such as solid storage and liquid slurry storage had much higher MCFs, at 20 and 30°C. However, these waste management methods are more prevalent in parts of the world where the average annual temperature is closer to 10°C. At that temperature, the MCF is negligible in all waste management systems. This study showed that the previously reported estimates of MCF for some waste management systems were higher than was actually the case. Consequently earlier estimates of the amount of methane generated globally from manures were higher than those found in this study.

Detrimental changes in body composition are often associated with declining levels of testosterone. Here we evaluated the notion that multipotent mesenchymal stem cells (MSC), that give rise to both fat and muscle tissue, can play a significant role to alter existing body composition in the adult. Transgenic mice with targeted androgen receptor (AR) overexpression in stem cells were employed. Wild-type littermate and AR-transgenic male and female mice were gonadectomized and left untreated for 2 months. After the hypogonadal period, mice were then treated with 5α-dihydrotestosterone (DHT) for 6 weeks. After orchidectomy (ORX), wild-type males have reduced lean mass and increased fat mass compared to shams. DHT treatment was beneficial to partially restore body composition. In wild-type females, ovariectomy (OVX) produced a similar change but there was no improvement with DHT. In targeted AR transgenic mice, DHT treatment increased lean and reduced fat mass to sham levels. In contrast to wild-type females, DHT treatment in female transgenic mice significantly ameliorated the increased fat and decreased lean mass changes that result after OVX. Our results indicate that DHT administration reduces fat mass and increases lean mass in wild-type males but not females, indicating that wild-type females are not as sensitive to androgen treatment. Because both male and female transgenic mice are more responsive than wild-type, results suggest that body composition remains linked to stem cell fate in the adult and that targeted androgen signaling in stem cells can play a significant role to reverse detrimental changes in body composition in both sexes.

The intact seed surface morphology in 45 taxa of Petunia sensu Jussieu native to South America (Petunia sensu Wijsman plus Calibrachoa) was compared under scanning electron microscopy. The existence of three groups of species, differentiated in terms of seed morphology, was revealed as follows: (1) all species of Petunia sensu Wijsman, having coarse wavy middle lamellae and anticlinal walls; (2) Calibrachoa parviflora and C. pygmaea, having fine wavy middle lamellae embedded in straight anticlinal walls; and (3) the other species of Calibrachoa, having straight middle lamellae and anticlinal walls. Close relationships between seed morphology and the other characteristics observable in the groups of species are discussed.

The endocrine disruption associated with alcohol (ethanol) abuse in both males and females is widely recognized. Ethanol intoxication and withdrawal in males results in significant reductions in androgen levels. Less is known about female alcoholics, and because the changes in testosterone concentrations remain controversial, we systematically characterized changes in sex steroids after chronic ethanol exposure and withdrawal in both sexes. Testosterone and 17β-estradiol concentrations were determined during chronic high intoxication, over a withdrawal time course, and following a period of abstinence using a genetic model of withdrawal vulnerability, the Withdrawal Seizure-Resistant (WSR) and -Prone (WSP) selected lines. In males, testosterone concentrations were significantly lower in intoxicated WSP mice after chronic ethanol exposure, and were dramatically and transiently reduced during the withdrawal period in both WSR and WSP lines. In contrast, testosterone levels were increased in intoxicated WSP females and in both WSR and WSP mice during withdrawal. Chronic ethanol exposure disrupted normal estrous cycling in WSP mice, associated with hyperandrogenemia while intoxicated. In abstinence, elevated testosterone was observed in both sexes but only in WSR mice. Estrogen levels were modestly reduced during withdrawal in both WSR and WSP lines, predominantly in males. These findings identify a mechanism based on altered androgen signaling that likely contributes to sex-specific responses during withdrawal. However, only WSR mice showed similar elevations in androgen long after withdrawal in both sexes, suggesting that genotype is an important determinant of steroid responses after abstinence. Increased androgen signaling in females as a consequence of chronic ethanol exposure may play an important and relatively uncharacterized role in sexually dimorphic responses to alcohol abuse.