Conventional expression studies quantify messenger RNA (mRNA) transcript levels gene-by-gene. We recently showed that protein expression is modulated at a global scale by amino acid availability, suggesting that mRNA expression levels might be equivalently affected. Through re-analysis of public transcriptomic datasets, it was confirmed that nucleobase supply interacts with the specific demands of mRNA A + U:C + G sequence composition to shape a global profile of expression, which can be quantified as a gradient of average expression change by average composition change. In mammals, each separate organ and cell-type displays a distinct baseline profile of global expression. These profiles can shift dynamically across the circadian day and the menstrual cycle. They are also significantly distorted by viral infection, multiple complex genetic disorders (including Alzheimer’s disease, schizophrenia, and autoimmune disorders), and after treatment with 115 of the 597 chemical entities analysed. These included known toxins and nucleobase analogues, but also many commonly prescribed medications such as antibiotics and proton pump inhibitors, thus revealing a new mechanism of drug action and side-effect. As well as key roles in disease susceptibility, mRNAs with extreme compositions are significantly over-represented in gene ontologies such as transcription and cell division, making these processes particularly sensitive to swings in global expression. This may permit efficient, en bloc transcriptional reprogramming of cell state through simple adjustment of nucleobase proportion and supply. It is also proposed that this mechanism helped mitigate the loss of essential amino acid synthesis in higher organisms. In summary, global expression regulation is invisible to conventional transcriptomic analysis, but its measurement allows a useful distinction between active, promoter-mediated gene expression changes and passive, cell state-dependent transcriptional competence. Linking cell metabolism directly to gene expression offers an entirely new perspective on evolution, disease aetiopathology (including gene x environment - GxE - interactions), and the nature of the pharmacological response.

The quantity of each protein in a cell only is only partially correlated with its gene transcription rate. Independent influences on protein synthesis levels include mRNA sequence motifs, amino acyl-tRNA synthesis levels, elongation factor action, and protein susceptibility to degradation. Here we report that the amino acid composition of a protein can also influence its expression level in two distinct ways. The nutritional classification of amino acids in animals reflects their potential for scarcity-essential amino acids (EAA) are reliant on dietary supply, non-essential amino acids (NEAA) from internal biosynthesis, and conditionally essential amino acids (CEAA) from both. Accessing public proteomic datasets, we demonstrate that a protein's CEAA sequence composition is inversely correlated with expression-a correlation enhanced during rapid cellular proliferation-suggesting CEAA availability can limit translation. Similarly, proteins with the most extreme compositions of EAA are generally reduced in abundance. These latter proteins participate in biological systems such as taste and food-seeking behaviour, oxidative phosphorylation, and chemokine function, and so linking their expression to EAA availability may act as a homeostatic response to malnutrition. Protein composition can also influence general human phenotypes and disease susceptibility: stature proteins are enriched in CEAAs, and a curated dataset of over 700 cancer proteins is significantly under-represented in EAAs. We also show that individual amino acids can influence protein expression across all kingdoms of life and that this effect appears to be rooted in the unchanging structural and mRNA encoding features of each amino acid. Species-specific environmental survival pathways are shown to be enriched in proteins with individual amino acid compositions favouring higher expression. These two forms of amino acid-driven protein expression regulation promise new insights into systems biology, evolutionary studies, experimental research design, and public health intervention.

Conventional expression studies quantify messenger RNA (mRNA) transcript levels gene-by-gene. We recently showed that protein expression is modulated at a global scale by amino acid availability, suggesting that mRNA expression levels might be similarly affected by nucleobase supply. Re-analysis of transcriptomic datasets confirmed that nucleobase supply and mRNA A+U:C+G sequence composition interact to shape a global profile of expression which can be represented by simple numerical outputs. In mammals, each separate organ and cell-type displays a distinct baseline profile of expression, influenced by differentiation state. Expression profiles shift dynamically across the circadian day and the menstrual cycle. They are also significantly distorted by viral infection, multiple complex genetic disorders (including Alzheimer's disease, schizophrenia, and autoimmune disorders), and after treatment with 115 of the 597 chemical entities analysed. These entities included known toxins, but also many commonly prescribed medications such as antibiotics and proton pump inhibitors, thus revealing a new mechanism of drug action and side-effect. A role for nucleobase supply is supported by the actions of nucleobase analogue treatments and by a model of the nucleobase metabolism disorder, Lesch-Nyhan syndrome. On the demand-side, mRNAs at compositional extremes are over-represented in key gene ontologies including transcription and cell division, making these processes particularly sensitive to swings in global expression. This permits efficient en bloc reprogramming of cell state through simple changes in nucleobase proportion and supply. It is also proposed that this mechanism helped mitigate the loss of essential amino acid synthesis in higher organisms. In summary, global expression regulation is invisible to conventional transcriptomic analysis, but its measurement allows a useful distinction between active, promoter-mediated gene expression changes and passive, cell state-dependent transcriptional competence. Linking metabolism directly to expression offers an entirely new perspective on evolution, disease aetiopathology (including GxE interactions), and the nature of the pharmacological response. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Type 2 diabetes mellitus is associated with increased aortic stiffness, a finding that predicts adverse cardiovascular events. Whether individuals with dysglycemia below the diagnostic threshold for diabetes have increased aortic stiffness has been less studied, particularly in Hispanic adults, a population sharing a disproportionate burden of dysglycemia. Hypothesis: We hypothesized that individuals with prediabetes would: (1) have increased aortic stiffness compared to normal glycemia, and (2) increased aortic stiffness would be associated with biomarkers of myocardial injury. Aims: We aimed to: (1) identify the relationship between aortic stiffness and glycemic status in a Hispanic cohort, and (2) determine the relationship between cardiac biomarkers and aortic stiffness. Methods: We prospectively measured carotid-femoral pulse wave velocity (PWV) and glycemic measures in 728 Mexican American adults in Starr County, Texas. Cardiovascular biomarkers [high-sensitivity troponin T (hs-TnT), galectin-3, and NT-proBNP] were measured in a subset (n=404). Multivariable models (using natural log PWV) were used to adjust for confounders. Results: 45% of participants had diabetes, and 28% had prediabetes. PWV increased across glycemic categories; median PWV (25 th , 75 th ) in diabetes was 11.7 m/sec (9.9, 13.7), prediabetes 9.9 m/sec (8.6, 11.1), and normal glycemia 8.7 m/sec (7.9, 9.9) (adjusted p-value <0.001 and 0.008, respectively). The figure demonstrates PWV stratified by age decade; PWV in prediabetes was similar to PWV in those without diabetes who were a decade younger. Median levels of hs-TnT, galectin-3, and NT-proBNP increased across quartiles of PWV (p trend <0.001 for all). Conclusion: In a cohort of Hispanic adults, increased aortic stiffness develops before the onset of diabetes and seen in those with pre-diabetes. Importantly, increased aortic stiffness is associated with biomarkers of cardiac injury.

Alzheimer’s disease is an irreversible neurodegenerative disease, which accounts for most dementia cases. Neuroinflammation is increasingly recognised for its roles in Alzheimer’s disease pathogenesis which, in part, links amyloid-beta to neuronal death. Neuroinflammatory signalling can be exhibited by neurons themselves, potentially leading to widespread neuronal cell death, although neuroinflammation is commonly associated with glial cells. The presence of the inflammasomes such as nucleotide-binding leucine-rich repeat receptors protein 1 in neurons accelerates amyloid-beta -induced neuroinflammation and has been shown to trigger neuronal pyroptosis in murine Alzheimer’s disease models. However, the pathways involved in amyloid-beta activation of inflammasomes have yet to be elucidated. In this study, a gene trap mutagenesis approach was utilised to resolve the genes functionally involved in inflammasome signalling within neurons, and the mechanism behind amyloid-beta-induced neuronal death. The results indicate that amyloid-beta significantly accelerated neuroinflammatory cell death in the presence of a primed inflammasome (the NLR family pyrin domain-containing 1). The mutagenesis screen discovered the atypical mitochondrial Ras homolog family member T1 as a significant contributor to amyloid-beta-induced inflammasome -mediated neuronal death. The mutagenesis screen also identified two genes involved in transforming growth factor beta signalling, namely Transforming Growth Factor Beta Receptor 1 and SNW domain containing 1. Additionally, a gene associated with cytoskeletal reorganisation, SLIT-ROBO Rho GTPase Activating Protein 3 was found to be neuroprotective. In conclusion, these genes could play important roles in inflammasome signalling in neurons, which makes them promising therapeutic targets for future drug development against neuroinflammation in Alzheimer’s disease.

Background Neuroinflammation is increasingly recognized for its roles in AD pathogenesis which, in part, links amyloid-beta (Aβ) to neuronal death. While commonly associated with glial cells, neurons themselves are able to participate in neuroinflammation signalling, potentially leading to widespread neuronal suicide. The presence of the inflammasomes such as NLRP1 in neurons accelerates Aβ-induced neuroinflammation and has been shown to trigger neuronal pyroptosis in murine AD models. However, the pathways involved in Aβ activation of inflammasomes has yet to be elucidated, especially in humans. In this study, we utilized a gene trap mutagenesis phenotypic screen approach to uncover the genes and biological pathways involved in inflammasome signalling in neurons and how it contributed to Aβ-induced neuronal death. Results Aβ significantly accelerated neuroinflammatory cell death in the presence of primed inflammasome. The gene trap mutagenesis screen discovered genes related to mitochondria function and TGF-β signalling as significant contributors to Aβ-induced inflammasome-driven neuronal death. Additionally, genes associated with cytoskeletal reorganization were found to confer neuroprotection. Conclusion Our data presents a list of potentially important components of inflammasome signalling in neurons which makes promising therapeutic targets for future drug development against neuroinflammation in AD.

The innate immune system and inflammatory response in the brain have critical impacts on the pathogenesis of many neurodegenerative diseases including Alzheimer's disease (AD). In the central nervous system (CNS), the innate immune response is primarily mediated by microglia. However, non-glial cells such as neurons could also partake in inflammatory response independently through inflammasome signalling. The NLR family pyrin domain-containing 1 (NLRP1) inflammasome in the CNS is primarily expressed by pyramidal neurons and oligodendrocytes. NLRP1 is activated in response to amyloid-β (Aβ) aggregates, and its activation subsequently cleaves caspase-1 into its active subunits. The activated caspase-1 proteolytically processes interleukin-1β (IL-1β) and interleukin-18 (IL-18) into maturation whilst co-ordinately triggers caspase-6 which is responsible for apoptosis and axonal degeneration. In addition, caspase-1 activation induces pyroptosis, an inflammatory form of programmed cell death. Studies in murine AD models indicate that the Nlrp1 inflammasome is indeed upregulated in AD and neuronal death is observed leading to cognitive decline. However, the mechanism of NLRP1 inflammasome activation in AD is particularly elusive, given its structural and functional complexities. In this review, we examine the implications of the human NLRP1 inflammasome and its signalling pathways in driving neuroinflammation in AD.

Diaminobutyric polypropylenimine (DAB) dendrimers have been shown to be highly efficient non-viral gene delivery systems for cancer therapy. However, their cytotoxicity currently limits their applications. To overcome this issue, PEGylation of DAB dendrimer, using various PEG molecular weights and dendrimer generations, has been attempted to decrease the cytotoxicity and enhance the DNA condensation, size and zeta potential, cellular uptake and transfection efficacy of these dendriplexes. Among all the PEGylated dendrimers synthesized, generation 3- and generation 4-DAB conjugated to low molecular weight PEG (2 kDa) at a dendrimer: DNA ratio of 20:1 and 10:1 resulted in an increase in gene expression on almost all tested cancer cells lines (by up to 3.2-fold compared to unmodified dendrimer in A431 cells). The highest level of β-galactosidase gene expression (10.07 × 10 −3 ± 0.09 × 10 −3 U/mL) was obtained following treatment of B16F10-Luc cells with G4-dendrimer PEGylated with PEG2K at a dendrimer: DNA ratio of 20:1. These delivery systems significantly decreased cytotoxicity on B16F10-Luc cells, by more than 3.4-fold compared to unmodified dendrimer. PEGylated generations 3- and 4-DAB dendrimers are therefore promising gene delivery systems for cancer therapy, combining low cytotoxicity and high transfection efficacy.

The possibility of using gene therapy for the treatment of brain diseases such as brain cancer, Alzheimer’s and Parkinson’s diseases, is currently hampered by the lack of gene delivery systems able to cross the blood-brain barrier and deliver DNA to the brain following intravenous administration. On the basis that lactoferrin can effectively reach the brain by using specific receptors for crossing the blood-brain barrier, we propose to investigate if a lactoferrin-bearing generation 3diaminobutyric polypropylenimine (DAB) dendrimer would allow the transport of plasmid DNA to the brain after intravenous administration. In this work, we demonstrated that the conjugation of lactoferrin to the dendrimer led to an enhanced DNA uptake by 2.1-fold in bEnd.3 murine brain capillary endothelial cells compared to the unmodified dendriplex in vitro. In vivo, the intravenous administration of lactoferrin-bearing DAB dendriplex resulted in a significantly increased gene expression in the brain, by more than 6.4-fold compared to that of DAB dendriplex, while decreasing gene expression in the lung and the kidneys. Gene expression in the brain was significantly higher than in any other major organs of the body. Lactoferrin-bearing generation 3 polypropylenimine dendrimer is therefore a highly promising delivery system for systemic gene delivery to the brain.

Existing criminal DNA profiling methods require prior inclusion of a profile within a criminal database. The ability to obtain descriptive information about an offender from DNA, regardless of database inclusion, would be of great use for investigators.

Lithium is the most successful mood stabilizer treatment for bipolar disorder. However, unlike conventional drugs that are designed to interact with a specific molecular target, the actions of lithium are distributed across many biological processes and pathways. Treatment response is subject to genetic variation between individuals and similar genetic variation may dictate susceptibility to side effects. Transcriptomic, genomic, and cell-model research strategies have all been deployed in the search for the genetic factors and biological systems that mediate the interaction between genetics and the therapeutic actions of lithium. In this review, recent findings from genome-wide studies and patient cell lines will be summarized and discussed from a standpoint that genuine progress is being made to define clinically useful mechanisms of this treatment, to place it in the context of bipolar disorder pathology, and to move towards a time when the prescription of lithium is targeted to those individuals who will derive the greatest benefit.

Mammalian ageing features biological attrition evident at cellular, genetic and epigenetic levels. Mutation of mitochondrial DNA, and nuclear DNA methylation changes are well established correlates of ageing. The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. Here we describe the bisulphite sequencing of mtDNA from 82 individuals aged 18-91 years. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse correlation with subject age suggesting their utility as epigenetic markers of ageing.

Human aging is associated with epigenetic modification of the genome. DNA methylation at cytosines appears currently as the best characterised modification that occurs during the mammalian lifetime. Such methylation changes at regulatory region can provide insights to track contributor age for criminal investigation.The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands in the promoter regions of a number of age related genes, of which four successfully showed changes across the lifespan (NPTX2, KCNQ1DN, GRIA2 and TRIM58). This technique is based on the detection of remaining input genome after digestion with a methylation-sensitive restriction enzyme. This study examined DNA specimens from 80 female subjects of various ages (18-91 years) obtained from blood, using primers designed to flank the studied gene loci. The data obtained from DNA methylation quantification showed successful discrimination among volunteered ages. Overall, the difference between predicted and real age was about 11 years and absolute mean differences (AMD) was only 7.2 years error. We suggest the EpiTect system can be used as fast and simple innovative tool in future forensic age estimation. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Although there is considerable genetic and pathologic evidence for an association between neuregulin 1 (NRG1) dysregulation and schizophrenia, the underlying molecular and cellular mechanisms remain unclear. Mutant mice containing disruption of the transmembrane (TM) domain of the NRG1 gene constitute a heuristic model for dysregulation of NRG1-ErbB4 signaling in schizophrenia. The present study focused on hitherto uncharacterized information processing phenotypes in this mutant line. Using a mass spectrometry-based metabolomics approach, we also quantified levels of unique metabolites in brain. Across 2 different sites and protocols, Nrg1 mutants demonstrated deficits in prepulse inhibition, a measure of sensorimotor gating, that is, disrupted in schizophrenia; these deficits were partially reversed by acute treatment with second, but not first-, generation antipsychotic drugs. However, Nrg1 mutants did not show a specific deficit in latent inhibition, a measure of selective attention that is also disrupted in schizophrenia. In contrast, in a "what-where-when" object recognition memory task, Nrg1 mutants displayed sex-specific (males only) disruption of "whatwhen" performance, indicative of impaired temporal aspects of episodic memory. Differential metabolomic profiling revealed that these behavioral phenotypes were accompanied, most prominently, by alterations in lipid metabolism pathways. This study is the first to associate these novel physiological mechanisms, previously independently identified as being abnormal in schizophrenia, with disruption of NRG1 function. These data suggest novel mechanisms by which compromised neuregulin function from birth might lead to schizophrenia-relevant behavioral changes in adulthood.

Metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed with schizophrenia, depression, bipolar disorder (SDB), diabetes, and controls. Chromatography on a ZICpHILIC column was used with detection by Orbitrap mass spectrometry. Data extraction was carried out with m/z Mine 2.14 with metabolite searching against an in-house database. There was no clear discrimination between the controls and the SDB samples on the basis of a principal components analysis (PCA) model of 755 identified or putatively identified metabolites. Orthogonal partial least square discriminant analysis (OPLSDA) produced clear separation between 17 of the controls and 19 of the SDB samples (R2CUM 0.976, Q2 0.671, p-value of the cross-validated ANOVA score 0.0024). The most important metabolites producing discrimination were the lipophilic amino acids leucine/isoleucine, proline, methionine, phenylalanine, and tyrosine; the neurotransmitters GABA and NAAG and sugar metabolites sorbitol, gluconic acid, xylitol, ribitol, arabinotol, and erythritol. Eight samples from diabetic brains were analysed, six of which grouped with the SDB samples without compromising the model (R2 CUM 0.850, Q2 CUM 0.534, p-value for cross-validated ANOVA score 0.00087). There appears on the basis of this small sample set to be some commonality between metabolic perturbations resulting from diabetes and from SDB.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, loss of the alveolar unit, and increased levels of oxidative damage to macromolecules, including DNA. 8‐oxoguanine (8‐OG) is the most common oxidative DNA lesion and its removal and repair is executed through the base excision repair pathway (BER). The purpose of this study was to determine whether enhancing the activity of the DNA glycosylase, Ogg1, would benefit epithelial cell health during an oxidative challenge. Paraquat, an herbicide that intercalates within the inner mitochondrial membrane was used in combination with A549 cells to produce excessive amounts of reactive oxygen species. Levels of 8‐OG were measured using immunofluorescence and single cell phenotypic analysis was undertaken using a high content imaging platform. Transduction of A549 cells with full length‐Ogg1 baculovirus lowered the maximal levels of 8‐OG in mtDNA by 200% as compared to null virus control cells. Conversely, administration of Ogg1 siRNA rendered the cells more vulnerable to paraquat, increasing 8‐OG content by almost 500%. Exemplars of small molecule Ogg1 activators identified through a high‐throughput‐screen, were shown to reduce paraquat‐induced 8‐OG formation by approximately 50%. Moreover, Ogg1 activators improved paraquat‐induced loss of mitochondrial membrane potential, while paraquat‐induced cytochrome c translocation to the nucleus was blocked. The paraquat‐stimulated decline in the cellular energy state, i.e., the ATP/ADP ratio, was prevented in the presence of the Ogg1 activators. Associated with the beneficial effects of the Ogg1 activators described above was the preservation of nuclear area. These data provide evidence of cytoprotection from oxidation of mtDNA when Ogg1 protein is increased or through allosteric activation of the protein, suggesting that the BER pathway may be a target for potential small molecule intervention in COPD. Support or Funding Information GlaxoSmithKline

Discovered in the remnants of waste at Soutra Aisle, Scotland's largest medieval hospital, the tubers of Lathyrus linifolius are thought to have hunger suppressing capabilities dating back to the 1700 s. Sir Robert Sibbald, founding member of the Royal College of Physicians, spoke about these tubers in his book “Provision for the Poor in time of Dearth and Scarcity” [1]. It is also thought that these tubers provided a boost of energy, and were used when soldiers were at war. The active component is suspected to be trans-anethole, although this has still to be confirmed. The aim of this study was to determine if the traditional usage of these tubers could help in the development of therapeutics for the future. Following initial in vitro cytotoxicity screening on various cell lines (HS27, PNT2, ZR75, U937, SHSY5Y, 3T3-L1, Hek293), and a preliminary feeding trial on Sprague Dawley rats (n = 2), it was found that both the solvent extracts and the full tuber (powdered) showed no toxicity at the concentrations tested (100 µg/ml-3.125 µg/ml in vitro; 42 mg/kg body weight, based on traditional dosage, in vivo). Subsequently, a larger in vivo feeding trial (n = 8 per group) was carried out to assess the appetite suppressing capabilities. This study showed no effect on body weight, food intake or water intake following treatment with the tuber in comparison to the control group (0.9% saline), and a second test group (treated with trans-anethole at 42 mg/kg BW). Appetite and hunger are complex processes controlled primarily in the hypothalamus [2]. To determine if the tuber has any effect on gene expression in the hypothalamus, the subjects were sacrificed and the hypothalamus dissected. RNA was extracted from the hypothalamus and RNA sequencing was carried out in a preliminary study (n = 1). The results evidently show that this tuber has an effect at RNA level, with 565 genes being upregulated (> 2-fold), and 642 genes being downregulated (> 2-fold) in comparison to the control group.

While schizophrenia and mental health are qualitatively distinct at the level of clinical presentation, the specific molecular signatures that underlie, or associate with, illness are not. Biomarker identification in schizophrenia is intended to offer a number of important benefits to patient well-being including prediction of future illness, diagnostic clarity and a level of disease description that would guide treatment choice. However, the choice of sample and form of analysis used to produce useful biomarkers is still uncertain. In this review, advances from recent studies spanning the technical spectrum are presented together with comment on their comparative strengths and weaknesses. To date, these studies have aided our understanding of the pathological processes associated with illness much more than they have provided robust biomarkers. A number of reasons for this observation are suggested, as are new strategies for the extraction of biomarkers from large '-omics' datasets.