The extrachromosomal circular DNA atlas of aged and young mouse brains.

Extrachromosomal circular DNA (eccDNA) refers to a distinct class of circular DNA molecules that exist independently from linear chromosomal DNA. Extensive evidence has firmly established the significant involvement of eccDNA in cancer initiation, progression, and evolutionary processes. However, the relationship between eccDNA and brain aging remains elusive. Here, we employed extrachromosomal circular DNA sequencing (Circle-seq) to generate a comprehensive dataset of eccDNA from six brain structures of both young and naturally-aged mice, including the olfactory bulb, medial prefrontal cortex, nucleus accumbens, caudate putamen, hippocampus, and cerebellum. Furthermore, through database annotation, we characterized the properties of mouse brain eccDNA, thereby gaining insights into the potential functions of eccDNA in the mouse brain. In conclusion, our study addresses a previously unexplored area by providing a comprehensive molecular characterization of eccDNA in brain tissues. The data presented in the study can be used as a fundamental resource to associate the molecular phenotypes of eccDNA with brain aging and gain deep insights into the biological role of eccDNA in mammalian brain aging.

Circle-seq based method for eccDNA synthesis and its application as a canonical promoter independent vector for robust microRNA overexpression.

Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathophysiologic states. The majority of circular DNAs detected by Circle-seq are small-size eccDNAs with enigmatic functions. One major technical hurdle is to synthesize eccDNA for functional identification. Here, we describe CAES (Circle-seq based Artificial EccDNA Synthesis), a promising and reliable method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2189 bp in size, were created utilizing the CAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by CAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that CAES-synthesized eccMIRs can express both miRNA-3p and - 5p molecules efficiently independent of a canonical promoter in human cell lines. Further assays proved that these eccMIRs were functional as they were able to repress the luciferase gene containing a miRNA-target sequence in the 3'UTR as well as the endogenous mRNA targets. Finally, kinetics study revealed that eccDNA exhibited a decay rate similar to the standard plasmids and linear DNA in cultured cells. Together, this study offers a rapid and convenient method for Circle-seq users to synthesize artificial eccDNAs. It also demonstrates the promising potential of eccMIR as a bacterial DNA-free vector for safe and robust miRNA overexpression in both basic research and therapeutic applications.

Ketogenic diet ameliorates attention deficit hyperactivity disorder in rats via regulating gut microbiota.

Attention deficit hyperactivity disorder (ADHD) is a common mental behavioral disorder in children. Alterations in gut microbiota composition are associated with neurological disorders. We aimed to investigate whether a ketogenic diet (KD) can be an alternative therapy for ADHD by altering the gut microbiota. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were randomly allocated to the normal diet (ND), methylphenidate (MPH), and KD groups. SHR in groups KD and MPH exhibited a significant increase in behavioral characteristics of ADHD, such as distance moved and immobility time. KD and MPH treatment led to a significant elevation in concentrations of 5-HT, AC, cAMP, and NE of brain tissue and the expression of DRD1, DAT, PKA, DARPP32, and cAMP at the protein level in WKY rats and SHR. KD and MPH significantly increased the richness and diversity of gut microbiota in SHR. The abundance of Ruminococcus_gauvreauii_group, Bacteroides, Bifidobacterium, and Blautia significantly increased, whereas that of Lactobacillus, Romboutsia, Facklamia, and Turicibacter significantly declined in the KD group compared with the ND group. The gut microbiota in the KD group of SHR mainly participated in amino acid metabolism- and sugar metabolism-related pathways. KD might alleviate behavioral disorders in ADHD by regulating gut microbiota. This study provides novel insights for the use of KD in treating ADHD.

A UPLC-MS/MS methodological approach for the analysis of 75 phenethylamines and their derivatives in hair.

A rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the targeted analysis of 75 phenethylamines and their derivatives from the hair matrix. The monitored classes of phenethylamines included the 2C series, D series, N-benzyl derivatives, mescaline-derived compounds, MDMA analogs, and benzodifurans. Approximately 20 mg of hair was weighed and pulverized with 0.1% formic acid in methanol by cryogenic grinding. After ultrasonication, centrifugation, and filtration, the supernatant was analyzed by LC-MS/MS operating in the scheduled multiple reaction monitoring mode. Phenethylamines and their derivatives were separated in 13 min on a biphenyl column (2.6 µm, 100 Å, 100 × 3.0 mm) using a gradient eluting mobile phase composed of 0.1% formic acid in water and acetonitrile. The developed and validated method showed good selectivity, sensitivity (LOD: 0.5-10 pg/mg and LOQ: 1-20 pg/mg), linearity (R2 > 0.997), accuracy and precision (< 20%), and stability. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of phenethylamines in hair from authentic forensic cases.

Effects of brain-derived neurotrophic factor and adeno-associated viral vector on morphine-induced condition through target concentration changes in the ventral tegmental area and nucleus accumbens.

Morphine remains the standard analgesic for severe pain. However, the clinical use of morphine is limited by the innate tendency of opiates to become addictive. Brain-derived neurotrophic factor (BDNF) is a growth factor that is protective against many mental disorders. This study aimed to evaluate the protective function of BDNF on morphine addiction based on the behavioural sensitisation (BS) model and assess potential changes in downstream molecular tropomyosin-related kinase receptor B (TrkB) and cyclic adenosine monophosphate response element binding protein (CREB) expression caused by overexpression of BDNF. We divided 64 male C57BL/6 J mice into saline, morphine, morphine plus adeno-associated viral vector (AAV), and morphine plus BDNF groups. After administering the treatments, behavioural tests were conducted during the development and expression phases of BS, followed by a western blot analysis. All data were analysed by one- or two-way analysis of variance. The overexpression of BDNF in the ventral tegmental area (VTA) caused by BDNF-AAV injection decreased the total distance of locomotion in mice who underwent morphine-induced BS and increased the concentrations of BDNF, TrkB, and CREB in the VTA and nucleus accumbens (NAc). BDNF exerts protective effects against morphine-induced BS by altering target gene expression in the VTA and NAc.

Brain-Derived Neurotrophic Factor Delivered Intranasally Relieves Post-Traumatic Stress Disorder Symptoms Caused by a Single Prolonged Stress in Rats.

INTRODUCTION: In our previous study, we successfully constructed the recombinant brain-derived neurotrophic factor (BDNF)-adeno-associated virus (AAV) modified by the influenza virus hemagglutinin-2 (HA2) and trans-transcriptional activator (TAT). BDNF-HA2TAT/AAV has been confirmed to have antidepression effects. BDNF-HA2TAT/AAV seems a promising therapy for post-traumatic stress disorder (PTSD) as the BDNF plays an important role in the function of the nervous system. However, the effects of BDNF-HA2TAT/AAV on PTSD caused by the single prolonged stress (SPS) model are unknown. METHODS: After the SPS model was established, BDNF-HA2TAT/AAV was administered (1 × 1011 vg per rat) through inhalation in the SPS + BDNF group for 2 weeks. Next, the rats underwent behavioral tests including an open-field test (OFT), elevated plus maze (EPM), and a forced swimming test (FST). Sera and hippocampi were obtained from the rats, and an enzyme-linked immune sorbent assay was performed to determine corticosterone concentration. Western blotting was conducted to determine BDNF, tyrosine kinase receptor B (TrkB), cAMP-response element-binding protein, and protein kinase B levels. RESULTS: BDNF-HA2TAT/AAV released anxiety-like and depression-like behaviors in OFT, EPM, and FST. BDNF-HA2TAT/AAV also results in high plasma concentrations of corticosterone, BDNF, and TrkB in the hippocampus. CONCLUSIONS: SPS is an excellent animal model to assess PTSD. BDNF-HA2TAT/AAV therapeutically effects PTSD caused by SPS, with changes seen in plasma corticosterone and BDNF-TrkB pathways within the hippocampus; therefore, BDNF-HA2TAT/AAV may be a promising treatment for patients with PTSD.

Forensic Analysis of 9 Poisoning Death Cases Caused by Oral Administration of Diphenidol.

OBJECTIVES: To analyze the characteristics of diphenidol poisoning cases and to provide clues and technical means for the identification of such cases. METHODS: Biological samples of 9 deaths caused by diphenidol poisoning were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the characteristics of these cases were analyzed retrospectively. RESULTS: Most of the deaths caused by diphenidol poisoning were young females. The dosage was between 60 and 300 tablets, and the mass concentration of diphenidol in the postmortem blood ranged from 0.87 to 99.00 µg/mL. There was no correlation between the dosage and the concentration of diphenidol in the blood. CONCLUSIONS: Diphenidol poisoning has the characteristics of high concealment and lethality. More attention should be paid to suicide cases, and diphenidol should be recommended as a routine detection item to avoid missing detection.

Characterized profiles of gut microbiota in morphine abstinence-induced depressive-like behavior.

Morphine is the most widely used analgesic for pain management worldwide. Abstinence of morphine could lead to neuropsychiatric symptoms, including depression. Gut microbiota is believed to contribute to the development of depression. However, the characteristics and potential role of gut microbiota in morphine abstinence-induced depression remain unclear. In the present study, we first established morphine abstinence-induced depressive behavior in mice. After dividing the mice into depressive and non-depressive groups, the gut microbiota of the mice was detected by 16S rRNA gene sequencing. The difference in the diversities and abundance of the gut microbiota were analyzed between groups. Then, the representative microbial markers that could distinguish each group were identified. In addition, gene function prediction of the operational taxonomic units (OTUs) with differential abundance between the depressive and non-depressive groups after morphine abstinence was conducted. Our results suggested that four weeks of abstinence from morphine did not change the richness of the gut microbiota. However, morphine abstinence influenced the gut microbial composition. Several specific genera of gut microbiota were identified as markers for each group. Interestingly, gene function prediction found that the fatty acid metabolism pathway was enriched in the OUTs in the depressive group compared with the non-depressive group after morphine abstinence. Our data suggested that gut microbiota dysbiosis was associated with morphine abstinence-induced depressive behavior, possibly by implicating the fatty acid metabolism pathway.

Bibliometrics Analysis in English and Chinese Literature on Drowning in Forensic Medicine from 1991 to 2020.

OBJECTIVES: To explore the research hotspots and development trends of the field of forensic drowning from 1991 to 2020 by bibliometrics methods. METHODS: Based on Web of Science, CNKI database, Wanfang Data knowledge service platform, python 3.9.2, CiteSpace 5.8.R3, Gephi 0.9.2, etc. were used to analyze the publishing trends, countries/regions, institutions, authors and topics of the study on drowning. RESULTS: A total of 631 English literature were obtained, including 59 articles from Chinese authors, and 386 Chinese literature were obtained. The Chinese and English journals with the largest number of related literatures were Chinese Journal of Forensic Science (80 articles) and Forensic Science International (106 articles), respectively. Japan published the most articles in English, and China ranked third. Osaka City Univ (Japan, 28 articles) published the most English articles, and Guangzhou Forens Sci Inst (China, 22 articles) ranked second. Among Chinese literature, Guangzhou Forens Sci Inst (32 articles) published the most. The topic analysis of Chinese and English literature showed that diatom examination, virtual autopsy, postmortem biochemical examination, the nature of death, and postmortem submersion interval were the hot spots of current research, but English literature had more studies on new technologies and methods, while Chinese literature was more inclined to practice, application and experience summary. CONCLUSIONS: The number of literature in forensic medicine on drowning is relatively stable. The scope of international and domestic collaborations in this field is still limited. The automated examination of diatoms, the establishment of diatom DNA barcodes and virtual autopsy will be the most important research hotspots in the coming period and are expected to achieve breakthroughs in drowning diagnosis, drowning location inference, postmortem submersion interval estimation, etc.

BDNF-AAV has protective effects on morphine-induced conditioned place preference through BDNF, TrkB, and CREB concentration changes in the VTA and NAc.

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophic factors that promotes the survival and protection of neurons in many disorders. The potential protective effect of BDNF and its mechanisms on morphine addiction are unclear. In this study, morphine-induced conditioned place preference (CPP) in mice was used to show the effect of BDNF on rewarding behavior. Western blot assays were used to determine the changes caused by BDNF, for example, changes in total BDNF, tropomyosin-related kinase receptor B (TrkB), and cAMP response element binding protein (CREB) in the ventral tegmental area (VTA) and nucleus accumbens (NAc). The results showed that the BDNF-adeno-associated viral vector (BDNF-AAV) injected in the VTA, attenuated morphine-induced CPP with synergistic changes in BDNF/TrkB/CREB concentrations in the VTA and NAc in the CPP acquisition and recurrence phases; however, the attenuation was lower in the extinction phase, with different changes in molecules downstream of the BDNF.

Tentative identification of in vitro metabolites of O-acetylpsilocin (psilacetin, 4-AcO-DMT) by UHPLC-Q-Orbitrap MS.

4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT, psilacetin, O-acetylpsilocin) is a synthetic tryptamine with psychedelic properties. Psilacetin may also act as precursor drug of psilocin, similar to psilocybin, but little is known about its metabolism. In this study, the phase I and phase II in vitro metabolism of 4-AcO-DMT was investigated with pooled human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. Fifteen metabolites were formed after incubation of pooled human liver microsomes with 4-AcO-DMT (12 phase I metabolites and 3 phase II metabolites). The proposed metabolite structures were based on accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydrolysis, hydroxylation, N-demethylation, oxidation, and conjugation with glucuronic acid. The hydrolysis metabolite was the most abundant compound. For the development of new methods for the identification of 4-AcO-DMT consumption, the beta-hydroxylation metabolite of 4-AcO-DMT (M2-1) is recommended as a biomarker. The data reported in this work might be applicable to metabolic transformation of 4-AcO-DMT in vivo and also forensically helpful.

Electrical lesion of bilateral ventrolateral orbital cortex impairs fear- and space-related learning and affects subsequent choice behavior.

OBJECTIVES: Although many studies have indicated that orbitofrontal cortex plays an important role in the learning and retrieval of memory and subsequent decision-making, the role of ventrolateral orbital cortex (VLO) still remains unclear, especially related to fear and space. METHODS: Four separate cohorts of rats were used in this study. After sham surgery and electrical lesion of bilateral VLO, four cohorts received active avoidance test, passive avoidance test, Morris water maze and T maze separately. RESULTS: Firstly, data shown that electrolytic lesions of bilateral VLO of Sprague-Dawley rats shortened the latency of rats to escape to darkroom in passive avoidance test. Besides, the damage of VLO also resulted in decrease of the number of active avoidance of rats from the third day during 5 consecutive days' training in active avoidance test. What's more, the impairment of VLO significantly shortened the exploring time in the target quadrant of rats in Morris water maze. Furthermore, VLO-lesions group shown lower correct alternation percentage than sham group in T maze. CONCLUSIONS: These results indicated that not only in the learning and retrieval of fear-related memory, VLO also plays an important role in the learning and retrieval of spatial-related memory guided by visual cues.

HINT1 deficiency in aged mice reduces anxiety-like and depression-like behaviours and enhances cognitive performances.

Histidine triad nucleotide-binding protein 1 (HINT1) is regarded as a haplo-insufficient tumour suppressor and is closely associated with many neuropsychiatric disorders, including major depressive disorders. In addition, HINT1 knockout (KO) mice exhibit anxiolytic-like behaviour, antidepression-like behaviour, and enhanced cognitive performance in several studies. However, it is still unclear whether aging contributes to these changes in the emotion and cognition of HINT1 KO mice. This study examined the role of aging in anxiety-like and depression-like behaviours and cognition behaviours in aged HINT1 KO mice compared with young HINT1 KO mice and their wild-type littermates, along with a number of molecular biological methods. In a battery of behavioural tests, aged wild-type mice showed increased anxiety-like and depression-like behaviours and decreased cognitive performance, along with lower expression levels of glutathione peroxidase, enhanced amount of malondialdehyde, and decreased expression levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus and PFC compared to young wild-type mice. HINT1 KO mice showed reduced anxiety-like and depression-like behaviours and enhanced cognitive performance compared to age-matched wild-type mice. In addition, HINT1 KO mice also showed increased GSH-Px and superoxide dismutase, and decreased malondialdehyde, together with enhanced BDNF and Trk-B expression in the hippocampus and PFC. However, when compared with young HINT1 KO mice, aged HINT1 KO mice did not show increased anxiety-like and depression-like behaviours. And there are no differences in the expression level of superoxide dismutase, malondialdehyde, BDNF, and Trk-B between aged and young HINT1 KO mice. In summary, HINT1 deficiency can counteract age-related emotion and cognition dysfunction.

Electrolytic lesions of the bilateral ventrolateral orbital cortex not only directly reduce depression-like behavior but also decreased desperate behavior induced by chronic unpredicted mild stress in rats.

BACKGROUND: Previous studies have revealed that ventrolateral orbital cortex (VLO) may play an important role in the regulation of emotional behavior. However, it is not known what effect VLO damage will have on emotion regulation. RESULTS: Data showed that damage of VLO increased the anxiety-like behavior in open field test and elevated plus maze, and decreased the depressive behavior in forced swimming test and learned helplessness test. Besides, the impulsive aggressive behaviors were also increased while the attack latency decreased after VLO lesion. What's more, damage of VLO decreased depressive behaviors induced by chronic unpredicted mild stress in rats. CONCLUSIONS: These results suggest that the integrity of VLO plays an important role in emotional regulation, and the damage of VLO may inhibit the development of depression-like behavior.

Characteristics of serum metabolites in sporadic amyotrophic lateral sclerosis patients based on gas chromatography-mass spectrometry.

To identify differential metabolites and metabolic pathways and provide guidance for the novel biomarkers for diagnosis and prognosis of amyotrophic lateral sclerosis (ALS). ALS patients and people without nervous diseases were recruited. Metabolomic analysis was performed using gas chromatography-mass spectrometry (GC/MS). The orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to identify differential metabolites. Kyoto Encyclopedia of Genes and Genomes and MetaboAnalyst were used to identify metabolic pathways. 75 metabolites were detected and aligned. The OPLS-DA showed the metabolomic profile of ALS patients and those in the fast-progression and slow-progression ALS groups differed from that of CTRL (p < 0.05). The levels of maltose, glyceric acid, lactic acid, beta-alanine, phosphoric acid, glutamic acid, ethanolamine and glycine in ALS were significantly higher, while 2,4,6-tri-tert-butylbenzenethiol was lower. Glycine, serine and threonine metabolism, D-glutamine and D-glutamate metabolism, alanine, aspartate, and glutamate metabolism, beta-alanine metabolism, and pyruvate metabolism were significantly altered metabolic pathways in ALS. ROC was used to discriminate ALS from CTRL with an AUC of 0.898 (p < 0.001) using 2,4,6-tri-tert-butylbenzenethiol, beta-alanine, glycine, and ethanolamine. The serum metabolites and metabolic pathways in ALS patients are significantly altered compared with CTRL. These findings may contribute to the early diagnosis of ALS.

HINT1 Is Involved in the Chronic Mild Stress Elicited Oxidative Stress and Apoptosis Through the PKC ε/ALDH-2/4HNE Pathway in Prefrontal Cortex of Rats.

Major depressive disorder (MDD) is a severe, highly heterogeneous, and life-threatening psychiatric disease which affects up to 21% of the population worldwide. A new hypothesis suggests that the mitochondrial dysfunction causing oxidative stress (OS) and dysregulation of apoptosis in brain might be one of the key pathophysiological factors in MDD. Histidine triad nucleotide binding protein 1 (HINT1), which was first supposed to be protein kinase C (PKC) inhibitor, has been gradually demonstrated to be involved in diverse neuropsychiatric diseases. It still remains elusive that how HINT1 involves in depression. The present study utilized a rat model exposed to chronic mild stress (CMS) to explore the involvement of HINT1 in depression. Face validity, construct validity and predictive validity of CMS model were comprehensive evaluated in this study. Behavioral tests including sucrose preference test, open field test, and elevated plus maze and forced swimming test revealed that stressed rats displayed elevated level of anxiety and depression compared with the controls. CMS rats showed a significant decrease of superoxide dismutase, and a marked increase malondialdehyde levels in prefrontal cortex (PFC). We also found the CMS rats had elevated expression of HINT1, decreased levels of phosphorylated-PKC ε and aldehyde dehydrogenase-two (ALDH-2), and accumulated 4-hydroxynonenal (4HNE) in PFC. Moreover, CMS increased the levels of cleaved caspase-3 and Bax, and decreased the level of Bcl-2 in PFC. The alterations in behavior and molecule were prevented by antidepressant venlafaxine. These results demonstrated that HINT1 was involved in the CMS elicited OS and apoptosis in PFC, probably through the PKC ε/ALDH-2/4HNE pathway. The results suggest that the suppression of HINT1 might have potential as a novel therapeutic strategy for depression.

Identifying metabolites of diphenidol by liquid chromatography-quadrupole/orbitrap mass spectrometry using rat liver microsomes, human blood, and urine samples.

In recent years, diphenidol [1,1-diphenyl-4-piperidino-1-butanol] has been one of the drugs that appears in suicide cases, but there are few research data on its metabolic pathways and main metabolites. Metabolite identification plays a key role in drug safety assessment and clinical application. In this study, in vivo and in vitro samples were analyzed with ultra-high-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. Structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, 10 Phase I metabolites and 5 glucuronated Phase II metabolites were found in a blood sample and a urine sample from authentic cases. Three other Phase I metabolites were identified in the rat liver microsomes incubation solution. The results showed that the main metabolic pathways of diphenidol in the human body include hydroxylation, oxidation, dehydration, N-dealkylation, methylation, and conjugation with glucuronic acid. This study preliminarily clarified the metabolic pathways and main metabolites of diphenidol. For the development of new methods for the identification of diphenidol consumption, we recommend using M2-2 as a marker of diphenidol entering the body. The results of this study provide a theoretical basis for the pharmacokinetics and forensic scientific research of diphenidol.

Effects of ketogenic diet on cognitive function in pentylenetetrazol-kindled rats.

Although the ketogenic diet (KD) is known to control seizures and improve cognition function in patients with drug-refractory epilepsy, the underlying mechanism remains unknown. In the present study, using pentylenetetrazol (PTZ)-induced and kindled rats, we found that KD significantly improved the impaired spatial reference memory of PTZ-kindled rats in the Morris water maze. To explore the mechanism underlying the action of KD in PTZ-kindled rats, quantitative real-time PCR (qRT-PCR) and immunohistochemical analysis were used to detect the expression of GluR1 and NR2B. The results showed that both the mRNA and protein expression of GluR1 and NR2B were significantly downregulated in the hippocampus of PTZ-kindled rats, while KD could observably improve both the mRNA and protein expression of GluR1 and NR2B in the hippocampus of PTZ-kindled rats. Additionally, KD improved the over-activated MAPK in PTZ-kindled rats, but not CAMKII, as detected by enzyme-linked immuno sorbent assay (ELISA), suggesting that the MAPK signaling pathway might be involved in the memory improvement of KD in PTZ-kindled rats. In conclusion, these results demonstrate that KD can indeed improve impaired spatial reference memory in PTZ-kindled rats, and KD can improve the expression of NR2B and GluR1.