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Approximately 1 in 4 people worldwide have non-alcoholic fatty liver disease (NAFLD); however, there are currently no medications to treat this condition. This study investigated the role of adiposity-associated orphan G protein-coupled receptor 75 (GPR75) in liver lipid accumulation. We profiled Gpr75 expression and report that it is most abundant in the brain. Next, we generated the first single-cell-level analysis of Gpr75 and identified a subpopulation co-expressed with key appetite-regulating hypothalamic neurons. CRISPR-Cas9-deleted Gpr75 mice fed a palatable western diet high in fat adjusted caloric intake to remain in energy balance, thereby preventing NAFLD. Consistent with mouse results, analysis of whole-exome sequencing data from 428,719 individuals (UK Biobank) revealed that variants in GPR75 are associated with a reduced likelihood of hepatic steatosis. Here, we provide a significant advance in understanding of the expression and function of GPR75, demonstrating that it is a promising pharmaceutical target for NAFLD treatment.
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Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Camundongos , Humanos , Masculino , Tecido Adiposo/metabolismo , Camundongos Knockout , Fígado/metabolismo , Feminino , AdiposidadeRESUMO
Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340-360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative-especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.
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Mitocôndrias , NAD , NAD/metabolismo , Anisotropia , Oxirredução , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
Cells emit light at ultra-low intensities: photons which are produced as by-products of cellular metabolism, distinct from other light emission processes such as delayed luminescence, bioluminescence, and chemiluminescence. The phenomenon is known by a large range of names, including, but not limited to, biophotons, biological autoluminescence, metabolic photon emission and ultraweak photon emission (UPE), the latter of which shall be used for the purposes of this review. It is worth noting that the photons when produced are neither 'weak' nor specifically biological in characteristics. Research of UPE has a long yet tattered past, historically hamstrung by a lack of technology sensitive enough to detect it. Today, as technology progresses rapidly, it is becoming easier to detect and image these photons, as well as to describe their function. In this brief review we will examine the history of UPE research, their proposed mechanism, possible biological role, the detection of the phenomenon, and the potential medical applications.
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Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase our quality of life and resilience to future pandemics. In the current study, we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p = 4.69 × 10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer, whereas the obesity-associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble-burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing. The subsequent observation that deletion of BE5.1 also significantly reduced BDNF-AS expression in mice suggests a novel mechanism in the regulation of BDNF expression common to mice and humans, which contributes to the modulation of mood and anxiety in both species.
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Ansiedade , Fator Neurotrófico Derivado do Encéfalo , Obesidade , Polimorfismo de Nucleotídeo Único , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ansiedade/genética , Ansiedade/metabolismo , Humanos , Camundongos , Obesidade/genética , Obesidade/metabolismo , Feminino , Masculino , Polimorfismo de Nucleotídeo Único/genética , Sequências Reguladoras de Ácido Nucleico/genética , Camundongos Endogâmicos C57BL , COVID-19 , Alelos , Hipotálamo/metabolismo , Estudo de Associação Genômica Ampla/métodos , Comportamento Animal/fisiologia , Tonsila do Cerebelo/metabolismo , Predisposição Genética para Doença/genéticaRESUMO
In this study, we have investigated a possible mechanism that enables CB1/M3 receptor cross-talk, using SH-SY5Y cells as a model system. Our results show that M3 receptor activation initiates signaling that rapidly upregulates the CNR1 gene, resulting in a greatly potentiated CB1 receptor response to agonists. Calcium homeostasis plays an essential intermediary role in this functional CB1/M3 receptor cross-talk. We show that M3 receptor-triggered calcium release greatly increases CB1 receptor expression via both transcriptional and translational activity, by enhancing CNR1 promoter activity. The co-expression of M3 and CB1 receptors in brain areas such as the nucleus accumbens and amygdala support the hypothesis that the altered synaptic plasticity observed after exposure to cannabinoids involves cross-talk with the M3 receptor subtype. In this context, M3 receptors and their interaction with the cannabinoid system at the transcriptional level represent a potential pharmacogenomic target not only for the develop of new drugs for addressing addiction and tolerance. but also to understand the mechanisms underpinning response stratification to cannabinoids.
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Canabinoides , Neuroblastoma , Humanos , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Cálcio/metabolismo , Canabinoides/farmacologia , Canabinoides/metabolismo , Sinalização do CálcioRESUMO
Alcohol use disorder (AUD) is one of the major causes of mortality and morbidity world-wide. It is estimated that 50% of the causes of AUD are heritable. Efforts to determine the genetic determinants governing AUD using genome wide association studies (GWAS) show that the most strongly associated SNPs occur within, or in the vicinity of, genes encoding enzymes that metabolise ethanol. However, these studies were not so conclusive in identifying the genes that influenced the choice to drink ethanol or why a proportion of the population become addicted. Most importantly, these studies also found that over 98% of the 1292 SNPs associated with AUD (p<1 × 10-6) were found outside of coding regions and within the poorly understood non-coding genome. Many years of study have shown that functional components of the non-coding genome include enigmatic enhancer elements whose biological role is to modulate levels of gene expression in specific cells, in specific amounts and in response to the correct stimuli. The current short review introduces the functional components of the non-coding genome, such as promoters and enhancers, and critically assesses the latest methods of identifying and characterising their context dependant roles in AUD and mental health disorders. We then go on to examine what is known about the roles of enhancers, such as the GAL5.1 enhancer, in alcohol intake and explore how enhancers are affected by polymorphic variation and epigenetic markers such as DNA-methylation and may influence susceptibility to AUD. The review finishes by discussing the future of AUD genetics and what technologies will need to be brought to bear to understand how genetic and environmentally induced changes in enhancer structure may contribute to the need to drink alcohol to excess.
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Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.
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Bancos de Espécimes Biológicos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Consumo de Bebidas Alcoólicas/genética , Animais , Ansiedade/genética , Etanol , Masculino , Camundongos , Reino UnidoRESUMO
Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.
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Alcoolismo/patologia , Ansiedade/patologia , Dieta Hiperlipídica , Elementos Facilitadores Genéticos/genética , 5-Metilcitosina/metabolismo , Alcoolismo/genética , Tonsila do Cerebelo/metabolismo , Animais , Ansiedade/genética , Linhagem Celular Tumoral , Metilação de DNA , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Epigênese Genética , Feminino , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C/química , Proteína Quinase C/metabolismoRESUMO
Sequencing of the human genome has permitted the development of genome-wide association studies (GWAS) to analyze the genetics of a number of complex disorders such as depression, anxiety and substance abuse. Thanks to their ability to analyze huge cohort sizes, these studies have successfully identified thousands of loci associated with a broad spectrum of complex diseases. Disconcertingly, the majority of these GWAS hits occur in non-coding regions of the genome, much of which controls the cell-type-specific expression of genes essential to health. In contrast to gene coding sequences, it is a challenge to understand the function of this non-coding regulatory genome using conventional biochemical techniques in cell lines. The current commentary scrutinizes the field of complex genetics from the standpoint of the large-scale whole-genome functional analysis of the promoters and cis-regulatory elements using chromatin markers. We contrast these large scale quantitative techniques against comparative genomics and in vivo analyses including CRISPR/CAS9 genome editing to determine the functional characteristics of these elements and to understand how polymorphic variation and epigenetic changes within these elements might contribute to complex disease and drug response. Most importantly, we suggest that, although the role of chromatin markers will continue to be important in identifying and characterizing enhancers, more emphasis must be placed on their analysis in relevant in-vivo models that take account of the appropriate cell-type-specific roles of these elements. It is hoped that offering these insights might refocus progress in analyzing the data tsunami of non-coding GWAS and whole-genome sequencing "hits" that threatens to overwhelm progress in the field.
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Elementos Facilitadores Genéticos , Predisposição Genética para Doença/genética , Edição de Genes , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Regiões Promotoras Genéticas , Sequenciamento Completo do GenomaRESUMO
Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (ß-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.
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Acetilglucosaminidase/metabolismo , Domínio Catalítico/genética , Quitina/metabolismo , Quitinases/metabolismo , Flavobacterium/enzimologia , Acetilglucosaminidase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/genética , Cristalografia por Raios X , Flavobacterium/genética , Flavobacterium/metabolismo , Modelos MolecularesRESUMO
A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα. L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα. However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.
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Ceramidas/biossíntese , Encefalite/metabolismo , Hipotálamo/metabolismo , Ácido Palmítico/administração & dosagem , Ácido Palmítico/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Encefalite/induzido quimicamente , Hipotálamo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-DawleyRESUMO
Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Toward addressing the hypothesis that genetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we characterized the effects of disease-associated allelic variation within a conserved regulatory sequence (ECR1) in CNR1 intron 2 that had previously been shown to modulate cannabinoid response, alcohol intake, and anxiety-like behavior. We used primary cell analysis of reporters carrying different allelic variants of the human ECR1 and found that human-specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in primary hippocampal cells than did the ancestral T-allele and demonstrated a differential response to CB1 agonism. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity and evidence that the ancestral t-allele variant of ECR1 interacted with higher affinity with the insulator binding factor CTCF. The cell-specific approaches used in our study represent an important step in gaining a mechanistic understanding of the roles of noncoding polymorphic variation in disease and in the increasingly important field of cannabinoid pharmacogenetics.
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Canabinoides/farmacologia , Sequência Conservada , Elementos Facilitadores Genéticos , Farmacogenética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Receptor CB1 de Canabinoide/genética , Células Cultivadas , Biologia Computacional/métodos , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Genes Reporter , Genes fos , Humanos , Especificidade de Órgãos/genética , Farmacogenética/métodosRESUMO
BACKGROUND: The rise in global obesity makes it crucial to understand how diet drives obesity-related health conditions, such as premature cognitive decline and Alzheimer's disease (AD). In AD hippocampal-dependent episodic memory is one of the first types of memory to be impaired. Previous studies have shown that in mice fed a high-fat diet (HFD) episodic memory is rapidly but reversibly impaired. METHODS: In this study we use hippocampal proteomics to investigate the effects of HFD in the hippocampus. Mice were fed either a low-fat diet (LFD) or HFD containing either 10% or 60% (Kcal) from fat for 3 days, 1 week or 2 weeks. One group of mice were fed the HFD for 1 week and then returned to the LFD for a further week. Primary hippocampal cultures were challenged with palmitic acid (PA), the most common long-chain saturated FA in the Western diet, and with the anti-inflammatory, n-3 polyunsaturated FA, docosahexaenoic acid (DHA), or a combination of the two to ascertain effects of these fatty acids on dendritic structure. RESULTS: HFD-induced changes occur in hippocampal proteins involved in metabolism, inflammation, cell stress, cell signalling, and the cytoskeleton after 3 days, 1 week and 2 weeks of HFD. Replacement of the HFD after 1 week by a low-fat diet (LFD) for a further week resulted in partial recovery of the hippocampal proteome. Microtubule-associated protein 2 (MAP2), one of the earliest proteins changed, was used to investigate the impact of fatty acids (FAs) on hippocampal neuronal morphology. PA challenge resulted in shorter and less arborised dendrites while DHA had no effect when applied alone but counteracted the effects of PA when FAs were used in combination. Dendritic morphology recovered when PA was removed from the cell culture media. CONCLUSION: This study provides evidence for the rapid and reversible effects of diet on the hippocampal proteome and the impact of PA and DHA on dendritic structure.
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The cannabinoid-1 receptor (CB1) plays a critical role in a number of biological processes including nutrient intake, addiction and anxiety-related behaviour. Numerous studies have shown that expression of the gene encoding CB1 (CNR1) is highly dynamic with changes in the tissue specific expression of CNR1 associated with brain homeostasis and disease progression. However, little is known of the mechanisms regulating this dynamic expression. To gain a better understanding of the genomic mechanisms modulating the expression of CNR1 in health and disease we characterised the role of a highly conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. We used CRISPR/CAS9 technology to disrupt ECR1 within the mouse genome. Disruption of ECR1 significantly reduced CNR1 expression in the hippocampus but not in the hypothalamus. These mice also displayed an altered sex-specific anxiety-related behavioural profile (open field test), reduced ethanol intake and a reduced hypothermic response following CB1 agonism. However, no significant changes in feeding patterns were detected. These data suggest that, whilst not all of the expression of CNR1 is modulated by ECR1, this highly conserved enhancer is required for appropriate physiological responses to a number of stimuli. The combination of comparative genomics and CRISPR/CAS9 disruption used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important first step in gaining a mechanistic understanding of cannabinoid regulatory pharmacogenetics.
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Consumo de Bebidas Alcoólicas/genética , Comportamento Aditivo/genética , Receptor CB1 de Canabinoide/genética , Animais , Ansiedade/genética , Transtornos de Ansiedade/genética , Encéfalo/metabolismo , Canabinoides/genética , Feminino , Predisposição Genética para Doença/genética , Genótipo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Íntrons/genética , Desequilíbrio de Ligação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/genética , Receptor CB1 de Canabinoide/metabolismoRESUMO
Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.
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Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Compostagem , Metagenoma , Sequência de Aminoácidos , Domínio Catalítico , Celulase/genética , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Hidrólise , Cinética , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato , TemperaturaRESUMO
Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Here we report atomic-resolution crystal structures of three such compounds in complexes with the cholera toxin. The structures give unprecedented atomic details of the molecular interactions and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their relatively easy synthesis and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics.
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Toxina da Cólera/química , Cólera/tratamento farmacológico , Enterotoxinas/química , Monossacarídeos/química , Toxinas Bacterianas/química , Sítios de Ligação , Cólera/microbiologia , Cristalografia por Raios X , Gangliosídeo G(M1)/química , Glicosídeos , Humanos , Modelos Moleculares , Monossacarídeos/antagonistas & inibidores , Ligação Proteica , Conformação Proteica/efeitos dos fármacosRESUMO
Neuropeptides and their receptors play a role in physiological responses such as appetite, stress and inflammatory pain. With neuropeptides having such diverse and important physiological roles, knocking-out the genes encoding them, their receptors, parts of their regulatory sequences, or reproducing disease associated polymorphic variants are important steps in studying neuropeptides and how they may contribute to disease. Previously, knock-outs were generated using methods such as targeted homologous recombination in embryonic stem cells but this method is costly and time-consuming. The CRISPR/Cas9 system has rapidly taken over the genome editing field and will advance our understanding of neuropeptide genes and their regulation. With CRISPR/Cas9 technology, the time and costs involved in producing transgenic animal models, is greatly reduced. In this review, we describe how the system can be used to manipulate genomic sequences by "knock-out" or "knock-in" mutations in cell lines or in animal models. We also discuss the specificity of the system and methods to limit off-target effects. When combined with the availability of genome sequences, CRISPR/Cas9 directed genome editing in vitro and in vivo, promises to provide a deeper understanding of the biology of the neuropeptides in health and disease than has ever been available before.
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Sistemas CRISPR-Cas , Expressão Gênica/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Neuropeptídeos/genética , Animais , Sistemas CRISPR-Cas/genética , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Humanos , Mutação/genéticaRESUMO
There can now be little doubt that the cis-regulatory genome represents the largest information source within the human genome essential for health. In addition to containing up to five times more information than the coding genome, the cis-regulatory genome also acts as a major reservoir of disease-associated polymorphic variation. The cis-regulatory genome, which is comprised of enhancers, silencers, promoters, and insulators, also acts as a major functional target for epigenetic modification including DNA methylation and chromatin modifications. These epigenetic modifications impact the ability of cis-regulatory sequences to maintain tissue-specific and inducible expression of genes that preserve health. There has been limited ability to identify and characterize the functional components of this huge and largely misunderstood part of the human genome that, for decades, was ignored as "Junk" DNA. In an attempt to address this deficit, the current chapter will first describe methods of identifying and characterizing functional elements of the cis-regulatory genome at a genome-wide level using databases such as ENCODE, the UCSC browser, and NCBI. We will then explore the databases on the UCSC genome browser, which provides access to DNA methylation and chromatin modification datasets. Finally, we will describe how we can superimpose the huge volume of study data contained in the NCBI archives onto that contained within the UCSC browser in order to glean relevant in vivo study data for any locus within the genome. An ability to access and utilize these information sources will become essential to informing the future design of experiments and subsequent determination of the role of epigenetics in health and disease and will form a critical step in our development of personalized medicine.
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Biologia Computacional/métodos , Metilação de DNA , Bases de Dados Genéticas , Epigenômica , Genoma Humano , Variação Genética , Humanos , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido NucleicoRESUMO
We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of "off target" effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome.