Habenular TCF7L2 links nicotine addiction to diabetes.

Diabetes is far more prevalent in smokers than non-smokers, but the underlying mechanisms of vulnerability are unknown. Here we show that the diabetes-associated gene Tcf7l2 is densely expressed in the medial habenula (mHb) region of the rodent brain, where it regulates the function of nicotinic acetylcholine receptors. Inhibition of TCF7L2 signalling in the mHb increases nicotine intake in mice and rats. Nicotine increases levels of blood glucose by TCF7L2-dependent stimulation of the mHb. Virus-tracing experiments identify a polysynaptic connection from the mHb to the pancreas, and wild-type rats with a history of nicotine consumption show increased circulating levels of glucagon and insulin, and diabetes-like dysregulation of blood glucose homeostasis. By contrast, mutant Tcf7l2 rats are resistant to these actions of nicotine. Our findings suggest that TCF7L2 regulates the stimulatory actions of nicotine on a habenula-pancreas axis that links the addictive properties of nicotine to its diabetes-promoting actions.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Conventional and multi-omics assessments of subacute inhalation toxicity due to propylene glycol and vegetable glycerin aerosol produced by electronic cigarettes.

Propylene glycol (PG) and vegetable glycerin (VG) are the most common solvents used in electronic cigarette liquids. No long-term inhalation toxicity assessments have been performed combining conventional and multi-omics approaches on the potential respiratory effects of the solvents in vivo. In this study, the systemic toxicity of aerosol generated from a ceramic heating coil-based e-cigarette was evaluated. First, the aerosol properties were characterized, including carbonyl emissions, the particle size distribution, and aerosol temperatures. To determine toxicological effects, rats were exposed, through their nose only, to filtered air or a propylene glycol (PG)/ glycerin (VG) (50:50, %W/W) aerosol mixture at the target concentration of 3 mg/L for six hours daily over a continuous 28-day period. Compared with the air group, female rats in the PG/VG group exhibited significantly lower body weights during both the exposure period and recovery period, and this was linked to a reduced food intake. Male rats in the PG/VG group also experienced a significant decline in body weight during the exposure period. Importantly, rats exposed to the PG/VG aerosol showed only minimal biological effects compared to those with only air exposure, with no signs of toxicity. Moreover, the transcriptomic, proteomic, and metabolomic analyses of the rat lung tissues following aerosol exposure revealed a series of candidate pathways linking aerosol inhalation to altered lung functions, especially the inflammatory response and disease. Dysregulated pathways of arachidonic acids, the neuroactive ligand-receptor interaction, and the hematopoietic cell lineage were revealed through integrated multi-omics analysis. Therefore, our integrated multi-omics approach offers novel systemic insights and early evidence of environmental-related health hazards associated with an e-cigarette aerosol using two carrier solvents in a rat model.

Central and peripheral actions of nicotine that influence blood glucose homeostasis and the development of diabetes.

Cigarette smoking has long been recognized as a risk factor for type 2 diabetes (T2D), although the precise causal mechanisms underlying this relationship remain poorly understood. Recent evidence suggests that nicotine, the primary reinforcing component in tobacco, may play a pivotal role in connecting cigarette smoking and T2D. Extensive research conducted in both humans and animals has demonstrated that nicotine can elevate blood glucose levels, disrupt glucose homeostasis, and induce insulin resistance. The review aims to elucidate the genetic variants of nicotinic acetylcholine receptors associated with diabetes risk and provide a comprehensive overview of the available data on the mechanisms through which nicotine influences blood glucose homeostasis and the development of diabetes. Here we emphasize the central and peripheral actions of nicotine on the release of glucoregulatory hormones, as well as its effects on glucose tolerance and insulin sensitivity. Notably, the central actions of nicotine within the brain, which encompass both insulin-dependent and independent mechanisms, are highlighted as potential targets for intervention strategies in diabetes management.

Nicotine transport across calu-3 cell monolayer: effect of nicotine salts and flavored e-liquids.

OBJECTIVE: This study aimed to investigate the transport capability of nicotine across Calu-3 cell monolayer in various nicotine forms, including nicotine freebase, nicotine salts, and flavored e-liquids with nicotine benzoate. SIGNIFICANCE: Nicotine is rapidly absorbed from the respiratory system into systemic circulation during e-cigarettes use. However, the mechanism of nicotine transport in the lung has not been well understood yet. This study may offer critical biological evidence and have implications for the use and regulation of e-cigarettes. METHODS: The viability of Calu-3 cells after administration of nicotine freebase, nicotine salts and representative e-liquid were evaluated using the MTT assay, and the integrity of the Calu-3 cell monolayer was evaluated by transepithelial electrical resistance measurement and morphological analysis. Further, the nicotine transport capacity across the Calu-3 cell monolayer in various formulations of nicotine was investigated by analysis of nicotine transport amount. RESULTS: The findings indicated that nicotine transport occurred passively and was time-dependent across the Calu-3cell monolayer. In addition, the nicotine transport was influenced by the type of nicotine salts and their respective pH value. The nicotine benzoate exhibited the highest apparent permeability coefficient (Papp), and higher nicotine-to-benzoic acid ratios led to higher Papp values. The addition of flavors to e-liquid resulted in increased Papp values, with the most significant increment being observed in tobacco-flavored e-liquid. CONCLUSIONS: In summary, the transport capability of nicotine across the Calu-3 cell monolayer was influenced by the pH values of nicotine salts and flavor additives in e-liquids.

The roles of the circadian hormone melatonin in drug addiction.

Given the devastating social and health consequences of drug addiction and the limitations of current treatments, a new strategy is needed. Circadian system disruptions are frequently associated with drug addiction. Correcting abnormal circadian rhythms and improving sleep quality may thus be beneficial in the treatment of patients with drug addiction. Melatonin, an essential circadian hormone that modulates the biological clock, has anti-inflammatory, analgesic, anti-depressive, and neuroprotective effects via gut microbiota regulation and epigenetic modifications. It has attracted scientists' attention as a potential solution to drug abuse. This review summarized scientific evidence on the roles of melatonin in substance use disorders at the cellular, circuitry, and system levels, and discussed its potential applications as an intervention strategy for drug addiction.

Evaluation of the inhalation toxicity of arecoline benzoate aerosol in rats.

Arecoline is a pharmacologically active alkaloid isolated from Areca catechu. There are no published data available regarding the inhalation toxicity of arecoline in animals. This study aimed to evaluate the inhalation toxicity of arecoline in vitro and in vivo. For this purpose, arecoline benzoate (ABA) salt was prepared to stabilize arecoline in an aerosol. The MTT assay determined the half-maximal inhibitory concentration values of ABA and arecoline in A549 cell proliferation to be 832 and 412 µg/ml, respectively. The toxicity of acute and subacute inhalation in Sprague-Dawley rats was evaluated using the guidelines of the Organization for Economic Cooperation and Development. For acute inhalation, the median lethal concentration value of ABA solvent was >5175 mg/m3 . After the exposure and during the recovery period, no treatment-related clinical signs were observed. In the 28-Day inhalation toxicity test, daily nose-only exposure to 2510 mg/m3 aerosol of the ABA solvent contained 75 mg/m3 ABA for male rats and 375 mg/m3 ABA for female rats, which caused no observed adverse effects, except for the decreased body weight gain in male rats exposed to 375 mg/m3 ABA. In this study, the no observed adverse effect level (NOAEL) for the 28-day repeated dose inhalation of ABA aerosol was calculated to be around 13 mg/kg/day for male rats and 68.8 mg/kg/day for female rats, respectively.

The Molecular Gut-Brain Axis in Early Brain Development.

Millions of nerves, immune factors, and hormones in the circulatory system connect the gut and the brain. In bidirectional communication, the gut microbiota play a crucial role in the gut-brain axis (GBA), wherein microbial metabolites of the gut microbiota regulate intestinal homeostasis, thereby influencing brain activity. Dynamic changes are observed in gut microbiota as well as during brain development. Altering the gut microbiota could serve as a therapeutic target for treating abnormalities associated with brain development. Neurophysiological development and immune regulatory disorders are affected by changes that occur in gut microbiota composition and function. The molecular aspects relevant to the GBA could help develop targeted therapies for neurodevelopmental diseases. Herein, we review the findings of recent studies on the role of the GBA in its underlying molecular mechanisms in the early stages of brain development. Furthermore, we discuss the bidirectional regulation of gut microbiota from mother to infant and the potential signaling pathways and roles of posttranscriptional modifications in brain functions. Our review summarizes the role of molecular GBA in early brain development and related disorders, providing cues for novel therapeutic targets.

Long noncoding RNA GM12371 acts as a transcriptional regulator of synapse function.

Despite the growing evidence suggesting that long noncoding RNAs (lncRNAs) are critical regulators of several biological processes, their functions in the nervous system remain elusive. We have identified an lncRNA, GM12371, in hippocampal neurons that is enriched in the nucleus and necessary for synaptic communication, synapse density, synapse morphology, and dendritic tree complexity. Mechanistically, GM12371 regulates the expression of several genes involved in neuronal development and differentiation, as well as expression of specific lncRNAs and their cognate mRNA targets. Furthermore, we find that cAMP-PKA signaling up-regulates the expression of GM12371 and that its expression is essential for the activity-dependent changes in synaptic transmission in hippocampal neurons. Taken together, our data establish a key role for GM12371 in regulating synapse function.

Profiling of key brain nuclei involved in CNS control of stress and glucose homeostasis.

Previous work have shown several key brain nuclei involved in acute psychological stress and glucose homeostasis. Acute stress influences glucose metabolism via released stress hormones by activating the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Little is known about the brain nuclei which response to peripheral glucose alteration are either abundant with glucosesensing neurons or the activations are secondary to stress. Here we profile and compare the brain nuclei that response to stress and glucose homeostasis in mouse models of acute restraint stress, glucose and 2-DG injections respectively. Our present work provide a comprehensive depiction on key brain nuclei involved in CNS control of stress and glucose homeostasis, which gives clue for functional identification of brain nuclei that regulate glucose homeostasis under stress.

Developing a sensitive HBV genotyping assay for HBV DNA suppressed patients using both DNA and RNA sequencing.

Hepatitis B virus (HBV) genotypes impact treatment outcomes and disease progression. The current genotyping methods have limitations in patients with low HBV viral load. In this study, a more sensitive assay has been developed for determining the HBV genotype in HBV DNA suppressed patients. Fifty-five serum samples from 55 chronic hepatitis B patients (HBeAg-, n = 20; HBeAg+, n = 35) across genotypes A to H with long-term nucleos(t)ide analogs (NAs) treatment were collected. All samples had HBV DNA less than 29 IU/mL. Total nucleic acid (viral DNA and RNA) was extracted and a 341 bp amplicon located at HBV S gene overlapping with reverse transcriptase domain of polymerase (pol/RT) was amplified via real time (RT)-nested polymerase chain reaction (PCR) followed by population sequencing. HBV genotype was determined by phylogenetic analysis. The assay successfully amplified HBV S/RT gene from 53 of 55 (96.4%) patient serum samples. Phylogenetic analysis demonstrated that the genotypes of all the 53 PCR positive samples matched the historical genotypes as determined by INNO-LiPA or RT sequence from the corresponding baseline samples. This assay was able to accurately determine HBV genotype irrespective of baseline genotype, HBeAg status, or duration of viral suppression. The ability to determine genotype in virally suppressed patients may facilitate the evaluation of novel treatment agents for HBV in this patient population.

Pulmonary pathology of early-phase COVID-19 pneumonia in a patient with a benign lung lesion.

AIMS: An ongoing outbreak of 2019 novel coronavirus (CoV) disease (COVID-19), caused by severe acute respiratory syndrome (SARS) CoV-2, has been spreading in multiple countries. One of the reasons for the rapid spread is that the virus can be transmitted from infected individuals without symptoms. Revealing the pathological features of early-phase COVID-19 pneumonia is important for understanding of its pathogenesis. The aim of this study was to explore the pulmonary pathology of early-phase COVID-19 pneumonia in a patient with a benign lung lesion. METHODS AND RESULTS: We analysed the pathological changes in lung tissue from a 55-year-old female patient with early-phase SARS-CoV-2 infection. In this case, right lower lobectomy was performed for a benign pulmonary nodule. Detailed clinical, laboratory and radiological data were also examined. This patient was confirmed to have preoperative SARS-CoV-2 infection by the use of real-time reverse transcription polymerase chain reaction and RNA in-situ hybridisation on surgically removed lung tissues. Histologically, COVID-19 pneumonia was characterised by exudative inflammation. The closer to the visceral pleura, the more severe the exudation of monocytes and lymphocytes. Perivascular inflammatory infiltration, intra-alveolar multinucleated giant cells, pneumocyte hyperplasia and intracytoplasmic viral-like inclusion bodies were seen. However, fibrinous exudate and hyaline membrane formation, which were typical pulmonary features of SARS pneumonia, were not evident in this case. Immunohistochemical staining results showed an abnormal accumulation of CD4+ helper T lymphocytes and CD163+ M2 macrophages in the lung tissue. CONCLUSION: The results highlighted the pulmonary pathological changes of early-phase SARS-CoV-2 infection, and suggested a role of immune dysfunction in the pathogenesis of COVID-19 pneumonia.

Substitution of O with a Single Au Atom as an Electron Acceptor in Al Oxide Clusters.

Al atoms generally adopt the +3 oxidation state and form stoichiometric oxides such as Al2O3 in the bulk phase. Among small cationic gas-phase clusters, near-stoichiometric clusters such as Al3O4+, Al3O5+, Al4O6+, Al4O7+, Al5O7+, and Al5O8+ have been readily generated in experimental studies. However, when a single Au atom was included in the clusters, oxygen-deficient clusters such as AuAl4O5+ were formed in high abundance; in these clusters, the Au atom accepted electron density from the Al atoms. The geometrical structures and atomic charges in the clusters suggest that a single Au atom can substitute for O atoms in Al oxide clusters. This propensity originates from the high electron and low oxygen affinities, which, together, constitute an unusual property of Au.

A novel data structure to support ultra-fast taxonomic classification of metagenomic sequences with k-mer signatures.

Motivation: Metagenomic read classification is a critical step in the identification and quantification of microbial species sampled by high-throughput sequencing. Although many algorithms have been developed to date, they suffer significant memory and/or computational costs. Due to the growing popularity of metagenomic data in both basic science and clinical applications, as well as the increasing volume of data being generated, efficient and accurate algorithms are in high demand. Results: We introduce MetaOthello, a probabilistic hashing classifier for metagenomic sequencing reads. The algorithm employs a novel data structure, called l-Othello, to support efficient querying of a taxon using its k-mer signatures. MetaOthello is an order-of-magnitude faster than the current state-of-the-art algorithms Kraken and Clark, and requires only one-third of the RAM. In comparison to Kaiju, a metagenomic classification tool using protein sequences instead of genomic sequences, MetaOthello is three times faster and exhibits 20-30% higher classification sensitivity. We report comparative analyses of both scalability and accuracy using a number of simulated and empirical datasets. Availability and implementation: MetaOthello is a stand-alone program implemented in C ++. The current version (1.0) is accessible via https://doi.org/10.5281/zenodo.808941. Contact: liuj@cs.uky.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Discerning novel splice junctions derived from RNA-seq alignment: a deep learning approach.

BACKGROUND: Exon splicing is a regulated cellular process in the transcription of protein-coding genes. Technological advancements and cost reductions in RNA sequencing have made quantitative and qualitative assessments of the transcriptome both possible and widely available. RNA-seq provides unprecedented resolution to identify gene structures and resolve the diversity of splicing variants. However, currently available ab initio aligners are vulnerable to spurious alignments due to random sequence matches and sample-reference genome discordance. As a consequence, a significant set of false positive exon junction predictions would be introduced, which will further confuse downstream analyses of splice variant discovery and abundance estimation. RESULTS: In this work, we present a deep learning based splice junction sequence classifier, named DeepSplice, which employs convolutional neural networks to classify candidate splice junctions. We show (I) DeepSplice outperforms state-of-the-art methods for splice site classification when applied to the popular benchmark dataset HS3D, (II) DeepSplice shows high accuracy for splice junction classification with GENCODE annotation, and (III) the application of DeepSplice to classify putative splice junctions generated by Rail-RNA alignment of 21,504 human RNA-seq data significantly reduces 43 million candidates into around 3 million highly confident novel splice junctions. CONCLUSIONS: A model inferred from the sequences of annotated exon junctions that can then classify splice junctions derived from primary RNA-seq data has been implemented. The performance of the model was evaluated and compared through comprehensive benchmarking and testing, indicating a reliable performance and gross usability for classifying novel splice junctions derived from RNA-seq alignment.

Activation of GSK-3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats.

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse-3 (GSK-3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK-3 by lateral ventricular infusion of wortmannin (WT) and GF-109203X (GFX), the inhibitors of phosphoinositol-3 kinase (PI3-K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor-κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK-3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK-3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.

New approach to capture and characterize synaptic proteome.

Little is known regarding the identity of the population of proteins that are transported and localized to synapses. Here we describe a new approach that involves the isolation and systematic proteomic characterization of molecular motor kinesins to identify the populations of proteins transported to synapses. We used this approach to identify and compare proteins transported to synapses by kinesin (Kif) complexes Kif5C and Kif3A in the mouse hippocampus and prefrontal cortex. Approximately 40-50% of the protein cargos identified in our proteomics analysis of kinesin complexes are known synaptic proteins. We also found that the identity of kinesins and where they are expressed determine what proteins they transport. Our results reveal a previously unappreciated role of kinesins in regulating the composition of synaptic proteome.

Distinct correlation network of clinical characteristics in suicide attempters having adolescent major depressive disorder with non-suicidal self-injury.

Suicidal behavior and non-suicidal self-injury (NSSI) are common in adolescent patients with major depressive disorder (MDD). Thus, delineating the unique characteristics of suicide attempters having adolescent MDD with NSSI is important for suicide prediction in the clinical setting. Here, we performed psychological and biochemical assessments of 130 youths having MDD with NSSI. Participants were divided into two groups according to the presence/absence of suicide attempts (SAs). Our results demonstrated that the age of suicide attempters is lower than that of non-attempters in participants having adolescent MDD with NSSI; suicide attempters had higher Barratt Impulsiveness Scale (BIS-11) impulsivity scores and lower serum CRP and cortisol levels than those having MDD with NSSI alone, suggesting levels of cortisol and CRP were inversely correlated with SAs in patients with adolescent MDD with NSSI. Furthermore, multivariate regression analysis revealed that NSSI frequency in the last month and CRP levels were suicidal ideation predictors in adolescent MDD with NSSI, which may indicate that the increased frequency of NSSI behavior is a potential risk factor for suicide. Additionally, we explored the correlation between psychological and blood biochemical indicators to distinguish suicide attempters among participants having adolescent MDD with NSSI and identified a unique correlation network that could serve as a marker for suicide attempters. Our research data further suggested a complex correlation between the psychological and behavioral indicators of impulsivity and anger. Therefore, our study findings may provide clues to identify good clinical warning signs for SA in patients with adolescent MDD with NSSI.

Histone deficiency and hypoacetylation in the aging retinal pigment epithelium.

Histones serve as a major carrier of epigenetic information in the form of post-translational modifications which are vital for controlling gene expression, maintaining cell identity, and ensuring proper cellular function. Loss of histones in the aging genome can drastically impact the epigenetic landscape of the cell leading to altered chromatin structure and changes in gene expression profiles. In this study, we investigated the impact of age-related changes on histone levels and histone acetylation in the retinal pigment epithelium (RPE) and retina of mice. We observed a global reduction of histones H1, H2A, H2B, H3, and H4 in aged RPE/choroid but not in the neural retina. Transcriptomic analyses revealed significant downregulation of histones in aged RPE/choroid including crucial elements of the histone locus body (HLB) complex involved in histone pre-mRNA processing. Knockdown of HINFP, a key HLB component, in human RPE cells induced histone loss, senescence, and the upregulation of senescence-associated secretory phenotype (SASP) markers. Replicative senescence and chronological aging in human RPE cells similarly resulted in progressive histone loss and acquisition of the SASP. Immunostaining of human retina sections revealed histone loss in RPE with age. Acetyl-histone profiling in aged mouse RPE/choroid revealed a specific molecular signature with loss of global acetyl-histone levels, including H3K14ac, H3K56ac, and H4K16ac marks. These findings strongly demonstrate histone loss as a unique feature of RPE aging and provide critical insights into the potential mechanisms linking histone dynamics, cellular senescence, and aging.

Hypothalamic CRF neurons facilitate brain reward function.

Aversive stimuli activate corticotropin-releasing factor (CRF)-expressing neurons in the paraventricular nucleus of hypothalamus (PVNCRF neurons) and other brain stress systems to facilitate avoidance behaviors. Appetitive stimuli also engage the brain stress systems, but their contributions to reward-related behaviors are less well understood. Here, we show that mice work vigorously to optically activate PVNCRF neurons in an operant chamber, indicating a reinforcing nature of these neurons. The reinforcing property of these neurons is not mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis. We found that PVNCRF neurons send direct projections to the ventral tegmental area (VTA), and selective activation of these projections induced robust self-stimulation behaviors, without activation of the HPA axis. Similar to the PVNCRF cell bodies, self-stimulation of PVNCRF-VTA projection was dramatically attenuated by systemic pretreatment of CRF receptor 1 or dopamine D1 receptor (D1R) antagonist and augmented by corticosterone synthesis inhibitor metyrapone, but not altered by dopamine D2 receptor (D2R) antagonist. Furthermore, we found that activation of PVNCRF-VTA projections increased c-Fos expression in the VTA dopamine neurons and rapidly triggered dopamine release in the nucleus accumbens (NAc), and microinfusion of D1R or D2R antagonist into the NAc decreased the self-stimulation of these projections. Together, our findings reveal an unappreciated role of PVNCRF neurons and their VTA projections in driving reward-related behaviors, independent of their core neuroendocrine functions. As activation of PVNCRF neurons is the final common path for many stress systems, our study suggests a novel mechanism underlying the positive reinforcing effect of stressful stimuli.