Comprehensive analysis of the gut microbiome and post-translational modifications elucidates the route involved in microbiota-host interactions.

The gut microbiome interacts with the host to maintain body homeostasis, with gut microbial dysbiosis implicated in many diseases. However, the underlying mechanisms of gut microbe regulation of host behavior and brain functions remain unclear. This study aimed to elucidate the influence of gut microbiota on brain functions via post-translational modification mechanisms in the presence or absence of bacteria without any stimulation. We conducted succinylome analysis of hippocampal proteins in germ-free (GF) and specific pathogen-free (SPF) mice and metagenomic analysis of feces from SPF mice. These results were integrated with previously reported hippocampal acetylome and phosphorylome data from the same batch of mice. Subsequent bioinformatics analyses revealed 584 succinylation sites on 455 proteins, including 54 up-regulated succinylation sites on 91 proteins and 99 down-regulated sites on 51 proteins in the GF mice compared to the SPF mice. We constructed a panoramic map of gut microbiota-regulated succinylation, acetylation, and phosphorylation, and identified cross-talk and relative independence between the different types of post-translational modifications in modulating complicated intracellular pathways. Pearson correlation analysis indicated that 13 taxa, predominantly belonging to the Bacteroidetes phylum, were correlated with the biological functions of post-translational modifications. Positive correlations between these taxa and succinylation and negative correlations between these taxa and acetylation were identified in the modulation of intracellular pathways. This study highlights the hippocampal physiological changes induced by the absence of gut microbiota, and proteomic quantification of succinylation, phosphorylation, and acetylation, contributing to our understanding of the role of the gut microbiome in brain function and behavioral phenotypes.

An optimized bioluminescent substrate for non-invasive imaging in the brain.

Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons.

Imaging of Evoked Cortical Depolarizations Using Either ASAP2s, or chi-VSFP, or Di-4-Anepps, or Autofluorescence Optical Signals.

BACKGROUND: Population voltage imaging is used for studying brain physiology and brain circuits. Using a genetically encoded voltage indicator (GEVI), "VSFP" or "ASAP2s", or a voltage-sensitive dye, Di-4-Anepps, we conducted population voltage imaging in brain slices. The resulting optical signals, optical local field potentials (LFPs), were used to evaluate the performances of the 3 voltage indicators. METHODS: In brain slices prepared from VSFP-transgenic or ASAP2s-transgenic mice, we performed multi-site optical imaging of evoked cortical depolarizations - compound excitatory postsynaptic potentials (cEPSPs). Optical signal amplitudes (ΔF/F) and cEPSP decay rates (OFF rates) were compared using analysis of variance (ANOVA) followed by unpaired Student's t test (31-104 data points per voltage indicator). RESULTS: The ASAP2s signal amplitude (ΔF/F) was on average 3 times greater than Di-4-Anepps, and 7 times greater than VSFP. The optical cEPSP decay (OFF rate) was the slowest in Di-4-Anepps and fastest in ASAP2s. When ASAP2s expression was weak, we observed slow, label-free (autofluorescence, metabolic) optical signals mixed into the ASAP2s traces. Fast hyperpolarizations, that typically follow depolarizing cortical transients (afterhyperpolarizations), were prominent in ASAP2s but not present in the VSFP and Di-4-Anepps experiments. CONCLUSIONS: Experimental applications for ASAP2s may potentially include systems neuroscience studies that require voltage indicators with large signal amplitude (ΔF/F), fast decay times (fast response time is needed for monitoring high frequency brain oscillations), and/or detection of brain patches in transiently hyperpolarized states (afterhyperpolarization).

Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

Integrated Metabolomics and Proteomics Analysis Revealed Second Messenger System Disturbance in Hippocampus of Chronic Social Defeat Stress Rat.

Depression is a common and disabling mental disorder characterized by high disability and mortality, but its physiopathology remains unclear. In this study, we combined a non-targeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis to elucidate metabolite and protein alterations in the hippocampus of rat after chronic social defeat stress (CSDS), an extensively used animal model of depression. Ingenuity pathway analysis (IPA) was conducted to integrate underlying relationships among differentially expressed metabolites and proteins. Twenty-five significantly different expressed metabolites and 234 differentially expressed proteins were identified between CSDS and control groups. IPA canonical pathways and network analyses revealed that intracellular second messenger/signal transduction cascades were most significantly altered in the hippocampus of CSDS rats, including cyclic adenosine monophosphate (cAMP), phosphoinositol, tyrosine kinase, and arachidonic acid systems. These results provide a better understanding of biological mechanisms underlying depression, and may help identify potential targets for novel antidepressants.

An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging.

Fluorescent indicators are used widely to visualize calcium dynamics downstream of membrane depolarization or G-protein-coupled receptor activation, but are poorly suited for non-invasive imaging in mammals. Here, we report a bright calcium-modulated bioluminescent indicator named Orange CaMBI (Orange Calcium-modulated Bioluminescent Indicator). Orange CaMBI reports calcium dynamics in single cells and, in the context of a transgenic mouse, reveals calcium oscillations in whole organs in an entirely non-invasive manner.

The correlation between oxidative stress level and intra-abdominal fat in obese males.

This study aims to investigate the correlation between oxidative stress and intra-abdominal fat (IAF) in obese young and middle-aged males.The present study included 136 male examinees in the Examination Center of the First Hospital of Qinhuangdao from October 10, 2015 to December 10, 2015. Then, clinical data, oxidative stress indices (8-iso-prostaglandin F2α [8-iso-PGF2α], malondialdehyde [MDA], and superoxide dismutase [SOD]), and IAF area were recorded. All subjects were assigned into 3 groups according to body mass index (BMI): obese group (BMI ≥ 28 kg/m, 43 subjects), overweight group (24 ≤ BMI < 28 kg/m, 46 subjects), and control group (BMI < 24 kg/m, 47 subjects). Then, statistical analysis was performed.There were significant differences in IAF area, leptin, adiponectin, 8-iso-PGF2α, MDA, SOD, fasting insulin (FINS), fasting blood glucose (FBG), and homeostasis model assessment-insulin resistance (HOMA-IR) among these 3 groups (P < .05). Male subjects in the obese group had higher leptin, 8-iso-PGF2α, MDA, FINS, and HOMA-IR levels, compared to subjects in the overweight and control groups. Furthermore, subjects in the overweight group had a larger IAF area and higher 8-iso-PGF2α, MDA, and FBG levels, when compared to controls. In addition, SOD was significantly lower in the obese and overweight groups than in the control group. However, there were no statistical differences in age, systolic and diastolic blood pressure, lipids, and islet ß-cell secretion function (homeostasis model assessment-ß) among these 3 groups (P ≥ .05). Moreover, the IAF area was positively correlated to 8-iso-PGF2α and MDA, and negatively correlated to SOD.Oxidative stress is significantly associated with the IAF area in obese males, and abdominal obesity could increase oxidative stress level and insulin resistance.

Differences in brain pathological changes between rotenone and 6-hydroxydopamine Parkinson's disease models.

Rotenone and 6-hydroxydopamine are two drugs commonly used to generate Parkinson's disease animal models. They not only achieve degenerative changes of dopaminergic neurons in the substantia nigra, but also satisfy the requirements for iron deposition. However, few studies have compared the characteristics of these two models by magnetic resonance imaging. In this study, rat models of Parkinson's disease were generated by injection of 3 µg rotenone or 10 µg 6-hydroxydopamine into the right substantia nigra. At 1, 2, 4, and 6 weeks after injection, coronal whole-brain T2-weighted imaging, transverse whole-brain T2-weighted imaging, and coronal diffusion tensor weighted imaging were conducted to measure fractional anisotropy and T2* values at the injury site. The fractional anisotropy value on the right side of the substantia nigra was remarkably lower at 6 weeks than at other time points in the rotenone group. In the 6-hydroxydopamine group, the fractional anisotropy value was decreased, but T2* values were increased on the right side of the substantia nigra at 1 week. Our findings confirm that the 6-hydroxydopamine-induced model is suitable for studying dopaminergic neurons over short periods, while the rotenone-induced model may be appropriate for studying the pathological and physiological processes of Parkinson's disease over long periods.

Plasma disturbance of phospholipid metabolism in major depressive disorder by integration of proteomics and metabolomics.

INTRODUCTION: Major depressive disorder (MDD) is a highly prevalent mental disorder affecting millions of people worldwide. However, a clear causative etiology of MDD remains unknown. In this study, we aimed to identify critical protein alterations in plasma from patients with MDD and integrate our proteomics and previous metabolomics data to reveal significantly perturbed pathways in MDD. An isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomics approach was conducted to compare plasma protein expression between patients with depression and healthy controls (CON). METHODS: For integrative analysis, Ingenuity Pathway Analysis software was used to analyze proteomics and metabolomics data and identify potential relationships among the differential proteins and metabolites. RESULTS: A total of 74 proteins were significantly changed in patients with depression compared with those in healthy CON. Bioinformatics analysis of differential proteins revealed significant alterations in lipid transport and metabolic function, including apolipoproteins (APOE, APOC4 and APOA5), and the serine protease inhibitor. According to canonical pathway analysis, the top five statistically significant pathways were related to lipid transport, inflammation and immunity. CONCLUSION: Causal network analysis by integrating differential proteins and metabolites suggested that the disturbance of phospholipid metabolism might promote the inflammation in the central nervous system.

Detecting dopaminergic neuronal degeneration using diffusion tensor imaging in a rotenone-induced rat model of Parkinson's disease: fractional anisotropy and mean diffusivity values.

Dopamine content in the basal ganglia is strongly associated with the degree of dopaminergic neuron loss in the substantia nigra pars compacta. Symptoms of Parkinson's disease might not arise until more than 50% of the substantia nigra pars compacta is lost and the dopamine content in the basal ganglia is reduced by more than 80%. Greater diagnostic sensitivity and specificity would allow earlier detection of Parkinson's disease. Diffusion tensor imaging is a recently developed magnetic resonance imaging technique that measures mean diffusivity and fractional anisotropy, and responds to changes in brain microstructure. When the microscopic barrier (including cell membranes, microtubules and other structures that interfere with the free diffusion of water) is destroyed and extracellular fluid volume accumulates, the mean diffusivity value increases; when the integrity of the microstructure (such as myelin) is destroyed, fractional anisotropy value decreases. However, there is no consensus as to whether these changes can reflect the early pathological alterations in Parkinson's disease. Here, we established a rat model of Parkinson's disease by injecting rotenone (or sunflower oil in controls) into the right substantia nigra. Diffusion tensor imaging results revealed that in the stages of disease, at 1, 2, 4, and 6 weeks after rotenone injection, fractional anisotropy value decreased, but mean diffusivity values increased in the right substantia nigra in the experimental group. Fractional anisotropy values were lower at 4 weeks than at 6 weeks in the right substantia nigra of rats from the experimental group. Mean diffusivity values were markedly greater at 1 week than at 6 weeks in the right corpus striatum of rats from the experimental group. These findings suggest that mean diffusivity and fractional anisotropy values in the brain of rat models of Parkinson's disease 4 weeks after model establishment can reflect early degeneration of dopaminergic neurons. The change in fractional anisotropy values after destruction of myelin integrity is likely to be of greater early diagnostic significance than the change in mean diffusivity values.

Social defeat stress causes depression-like behavior with metabolite changes in the prefrontal cortex of rats.

Major depressive disorder is a serious mental disorder with high morbidity and mortality. The role of social stress in the development of depression remains unclear. Here, we used the social defeat stress paradigm to induce depression-like behavior in rats, then evaluated the behavior of the rats and measured metabolic changes in the prefrontal cortex using gas chromatography-mass spectrometry. Within the first week after the social defeat procedure, the sucrose preference test (SPT), open field test (OFT), elevated plus maze (EPM) and forced swim test (FST) were conducted to examine the depressive-like and anxiety-like behaviors. For our metabolite analysis, multivariate statistics were applied to observe the distribution of all samples and to differentiate the socially defeated group from the control group. Ingenuity pathway analysis was used to find the potential relationships among the differential metabolites. In the OFT and EPM, there were no significant differences between the two experimental groups. In the SPT and FST, socially defeated rats showed less sucrose intake and longer immobility time compared with control rats. Metabolic profiling identified 25 significant variables with good predictability. Ingenuity pathways analysis revealed that "Hereditary Disorder, Neurological Disease, Lipid Metabolism" was the most significantly altered network. Stress-induced alterations of low molecular weight metabolites were observed in the prefrontal cortex of rats. Particularly, lipid metabolism, amino acid metabolism, and energy metabolism were significantly perturbed. The results of this study suggest that repeated social defeat can lead to metabolic changes and depression-like behavior in rats.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD).

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

Synthesis and antitumor activity of novel N-substituted carbazole imidazolium salt derivatives.

A series of novel N-substituted carbazole imidazolium salt derivatives has been prepared and investigated for their cytotoxic activity against five human tumor cell lines by MTS assay. The results indicated that the existence of 5,6-dimethyl-benzimidazole ring, substitution of the imidazolyl-3-position with a 2-bromobenzyl or naphthylacyl group, as well as alkyl chain length between carbazole and imidazole ring were important for the antitumor activity. Compound 61, bearing a 2-bromobenzyl substituent at position-3 of the 5,6-dimethyl-benzimidazole, showed powerful inhibitory activities and was more selective to HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values 0.51-2.48 µM. Mechanism of action studies revealed that this new compound could remarkably induce cell cycle arrest and apoptosis in SMMC-7721 cells. This work provides alternative novel way for future drug development based on carbazole and imidazolium salt scaffolds.

Non-high-density lipoprotein cholesterol and other risk factors of mild cognitive impairment among Chinese type 2 diabetic patients.

OBJECTIVE: To identify some risk factors of MCI among patients with type 2 diabetes(T2DM) and to find if there is any correlation between these factors and the degree of cognitive decline. METHODS: A total of 155 patients with T2DM referred to the Department of Endocrinology at First Hospital of Qinhuangdao were enrolled. To assess MCI the Montreal Cognitive Assessment (MoCA) scoring system was used. There were 66 patients with MCI and 89 patients without MCI (control). HbAlc, blood lipid, liver and renal functions were measured in all subjects. RESULTS: Compared with the control group, type 2 diabetic patients with MCI had a longer duration of diabetes; higher non-high-density lipoprotein cholesterol (non-HDL-C), triglycerides, total cholesterol, HbA1c, and BMI; and lower high-density lipoprotein cholesterol (HDL-C) (P<0.05). The rates of patients with a history of habitual light-to-moderate alcohol consumption, a high proportion of Mediterranean-type diet, and regular physical activity were lower; and the rate of current smoking was higher in type 2 diabetic patients with MCI than the control group (P<0.05). Among patients with MCI, the results indicated that MoCA score was negatively correlated with non-HDL-C (r=-0.761 P<0.001). CONCLUSIONS: Our results suggest that non-HDL-C can act as a readily available method for estimating risk of MCI in Chinese type 2 diabetic patient in routine clinical practice. Good lifestyle likely reduces MCI risk in diabetic patients.

Synthesis and antitumor activities of novel dibenzo[b,d]furan-imidazole hybrid compounds.

A series of novel hybrid compounds between dibenzo[b,d]furan and imidazole has been prepared and evaluated in vitro against a panel of human tumor cell lines. The results suggest that the existence of benzimidazole ring, and the substitution of the imidazolyl-3-position with a naphthylacyl or 4-methoxyphenacyl group, were vital for modulating cytotoxic activity. In particular, hybrid compound 60 was found to be the most potent derivatives against all of human tumor cell lines investigated, while compound 49 was found to be more selective against breast carcinoma (MCF-7) and myeloid liver carcinoma (SMMC-7721). Compound 60 can induce the G1 phase cell cycle arrest and apoptosis in SMMC-7721 cells.

Design, synthesis and biological evaluation of novel hybrid compounds of imidazole scaffold-based 2-benzylbenzofuran as potent anticancer agents.

A series of novel hybrid compounds between 2-benzylbenzofuran and imidazole has been prepared and evaluated in vitro against a panel of human tumor cell lines. The results suggest that the existence of benzimidazole ring and substitution of the imidazolyl-3-position with a naphthylacyl or 4-methoxyphenacyl group were vital for modulating cytotoxic activity. In particular, hybrid compounds 46 and 47 were found to be the most potent derivatives against 5 strains human tumor cell lines and more active than cisplatin (DDP), and exhibited cytotoxic activities selectively against breast carcinoma (MCF-7) and myeloid liver carcinoma (SMMC-7721), respectively.

Application of T2 measurement on gradient echo T2-weighted imaging in differential diagnosis of intracranial hemorrhage and calcification.

BACKGROUND: Differential diagnosis of intracranial hemorrhage and calcification is a common problem encountered in clinical imaging diagnosis. The purpose of this study was to investigate the feasibility of T2 measurement on gradient echo (GRE) T2-weighted imaging (T2WI) in differential diagnosis of intracranial hemorrhage and calcification. METHODS: Thirty-eight hemorrhagic foci in 18 patients and 11 calcification foci in seven patients were included in this study. The diagnosis of hemorrhage and calcification was confirmed in all cases with enhanced T2 weighted angiography (ESWAN) magnetic resonance imaging (MRI) and CT respectively. The significance for the difference of T2 value between the central and peripheral areas of hemorrhage and calcification lesions was tested with univariate analysis of variance. RESULTS: The detection rate of GRE T2 WI on intracranial hemorrhage was 1.9-fold higher than that of CT, especially for the hemorrhage in the brainstem and cerebellum. However, GRE T2WI was far less sensitive to calcification than CT. There was a significant difference in the T2 value between the central area of hemorrhage and calcification (P < 0.001), though no difference in the T2 value was obtained between the peripheral area of hemorrhage and calcification (P > 0.05). CONCLUSIONS: Quantitative measurement of T2 value on GRE T2 WI with a single MRI examination provides a fast, convenient, and effective means in differential diagnosis between intracranial hemorrhage and calcification, which may thus reduce the medical cost and save precious time for clinical management.