A UPLC-MS/MS method for simultaneous determination of arachidonic acid, stearic acid, and related endocannabinoids in human plasma.

Endocannabinoids (eCBs) exert considerable influence over energy metabolism, lipid metabolism, and glucose metabolism within the human body. Among the most biologically active cannabinoids identified thus far are 2-arachidonoylglycerol (2-AG), arachidonoyl ethanolamide (AEA), 1-stearoylglycerol (1-SRG), and stearoyl ethanolamide (SEA), which are derived from arachidonic acid (AA) and stearic acid (SA). However, despite the unique in bioactivities exhibited by eCBs, their determination in plasma has been hindered by the lack of sensitive analytical methods. The aim of this study was to develop and validate a highly sensitive and rapid method using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for accurate measurement of AEA, SEA, 2-AG, 1-SRG, AA, and SA levels in human plasma samples. Sample preparation involved a protein precipitation method and a methyl tert-butyl ether liquid-liquid extraction method. Chromatographic separation was accomplished by utilizing an ACQUITY UPLC BEH C8 column with a mobile phase of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid, flowing at a rate of 0.35 mL/min. AA-d8, 2-AG-d5, and AEA-d8 were selected as deuterated internal standards. The analytes were determined with MRM in both positive and negative ion mode. The lower limit of quantification ranged from 0.1 to 400 ng/mL, and the correlation coefficient (R2) was >0.99. Inter-day and intra-day precision exhibited values of 0.55-13.29% and 0.62%-13.90%, respectively. Recovery and matrix effect were within the range of 77.7%-109.7%, and 90.0%-113.5%, respectively. Stability tests confirmed the acceptability of all analytes. To demonstrate the effectiveness of the approach, it was implemented to assess and compare plasma samples from healthy volunteers (n = 49) and individuals with non-alcoholic fatty liver disease (NAFLD) (n = 62). The study revealed significant differences in AEA, SEA, AA, and SA levels between the two groups.

Palmatine inhibits expression fat mass and obesity associated protein (FTO) and exhibits a curative effect in dextran sulfate sodium (DSS)-induced experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease whose pathogenesis and mechanisms have not been fully described. The m6A methylation modification is a general mRNA modification in mammalian cells and is closely associated with the onset and progression of inflammatory bowel disease (IBD). Palmatine (PAL) is a biologically active alkaloid with anti-inflammatory and protective effects in animal models of colitis. Accordingly, we examined the role of PAL on colitis by regulating N6-methyladenosine (m6A) methylation. METHODS: A rat experimental colitis model was established by 5 % dextran sulfate sodium (DSS) in drinking water for seven days, then PAL treatment was administered for seven days. The colonic tissue pathology was assessed using hematoxylin-eosin (HE) and disease activity index (DAI). In in vitro studies, a human, spontaneously immortalized non-cancerous colon mucosal epithelial cell line (NCM460) was exposed to 2 % DSS and treated with PAL and cell viability was assayed using Cell Counting Kit-8 (CCK-8). The levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA) kits. The level of Zonula occludens-1 (ZO-1) was dectected by immunofluorescence. Transepithelial electrical resistance (TEER) of cells was also assessed. The methyltransferase-like 3 (METTL3), METTL14, AlkB homologate 5 (ALKBH5), and fat mass and obesity-associated protein (FTO) expression levels were assessed by western blotting. The localized expression of m6A was measured by immunofluorescence. RESULTS: PAL significantly prevented bodyweight loss and shortening of the colon in experimental colitis rats, as well as decreasing the DAI and histological damage scores. Furthermore, PAL inhibited the levels of inflammatory factors (TNF-α, IL-6, IL-8, and IL-1ß) in both DSS treated rats and NCM460 cells. In addition, PAL enhanced the expression level of ZO-1, and increased the transepithelial electrical resistance to repaire intestinal barrier dysfunction. Colitis occurred due to decreased m6A levels, and the increased FTO expression led to a colitis phenotype. PAL markedly enhanced the METTL3 and METTL14 expression levels while decreasing ALKBH5 and FTO expression levels. CONCLUSIONS: The findings demonstrated that PAL improved DSS-induced experimental colitis. This effect was associated with inhibiting FTO expression and regulating m6A methylation.

The aldehyde dehydrogenase 2 rs671 variant enhances amyloid ß pathology.

In the ALDH2 rs671 variant, a guanine changes to an adenine, resulting in a dramatic decrease in the catalytic activity of the enzyme. Population-based data are contradictory about whether this variant increases the risk of Alzheimer's disease. In East Asian populations, the prevalence of the ALDH2 rs671 variant is 30-50%, making the National Human Brain Bank for Development and Function (the largest brain bank in East Asia) an important resource to explore the link between the ALDH2 rs671 polymorphism and Alzheimer's disease pathology. Here, using 469 postmortem brains, we find that while the ALDH2 rs671 variant is associated with increased plaque deposits and a higher Aß40/42 ratio, it is not an independent risk factor for Alzheimer's disease. Mechanistically, we show that lower ALDH2 activity leads to 4-HNE accumulation in the brain. The (R)-4-HNE enantiomer adducts to residue Lys53 of C99, favoring Aß40 generation in the Golgi apparatus. Decreased ALDH2 activity also lowers inflammatory factor secretion, as well as amyloid ß phagocytosis and spread in brains of patients with Alzheimer's disease. We thus define the relationship between the ALDH2 rs671 polymorphism and amyloid ß pathology, and find that ALDH2 rs671 is a key regulator of Aß40 or Aß42 generation.

Reduced neurogenesis in human hippocampus with Alzheimer's disease.

Adult hippocampal neurogenesis (AHN), essential for the plasticity of hippocampal structure and function, may be disrupted in Alzheimer's disease (AD). However, the relationship between the changes in AHN and AD-related pathology in humans remains uncertain. By utilizing advanced immunostaining techniques, we could identify multiple biomarkers representing different stages of AHN in postmortem human hippocampal tissue that exhibited various AD-related neuropathological changes. In this study, we observed a significant presence of neurogenic cells in the hippocampus's dentate gyrus (DG) region in 30 individuals, including 14 individuals diagnosed with AD-related neuropathological changes and the remaining 16 individuals without any neurological diseases. Further investigation revealed that patients with AD exhibited pronounced astrogliosis and reduced neurogenesis. Specifically, the number of neuroblasts, immature and early mature granule cells decreased significantly as AD advanced. Although the number of neural stem cells (NSCs) remained unchanged in AD patients compared with mentally healthy individuals, they tended to be more quiescent state regulated by Notch and bone morphogenetic protein (BMP) signaling pathways. These abnormalities were strongly associated with the neuropathological alterations in AD patients. These research findings provide potential insights into the underlying mechanisms that underpin the pathogenesis of AD.

Arteriolosclerosis differs from venular collagenosis in relation to cerebrovascular parenchymal damages: an autopsy-based study.

BACKGROUND AND PURPOSE: Cerebrovascular parenchymal damage is prevalent in ageing brains; however, its vascular aetiology has not been fully elucidated. In addition to the underlying role of sclerotic arterioles, the correlation between collagenised venules has not been clarified. Here, we aimed to investigate the associations between microvascular injuries, including arteriolosclerosis and venular collagenosis, and related parenchymal damages in ageing brains, to investigate the underlying correlations. METHODS: We evaluated arteriolosclerosis and venular collagenosis in 7 regions from 27 autopsy cases with no history of stroke or brain tumour. The correlations between the ratio of arteriolosclerosis, venular collagenosis and the severity of cerebrovascular parenchymal damage, including lacunes, microinfarcts, myelin loss, and parenchymal and perivascular haemosiderin deposits, were assessed. RESULTS: Arteriolosclerosis and venular collagenosis became more evident with age. Arteriolosclerosis was associated with lacunes (p=0.004) and brain parenchymal haemosiderin deposits in the superior frontal cortex (p=0.024) but not with leukoaraiosis severity. Venular collagenosis was not associated with the number of lacunes or haemosiderin, while white matter generally became paler with severe venular collagenosis in the periventricular (ß=-0.430, p=0.028) and deep white matter (ß=-0.437, p=0.025). CONCLUSION: Our findings imply an important role for venular lesions in relation to microvessel-related parenchymal damage which is different from that for arteriolosclerosis. Different underlying mechanisms of both cerebral arterioles and venules require further investigation.

[Lipid-lowering effect of drug pair Scutellariae Radix-Coptidis Rhizoma based on lipomics].

This study investigated the mechanism of action of Scutellariae Radix-Coptidis Rhizoma(SR-CR) in intervening in non-alcoholic fatty liver disease(NAFLD) in rats based on lipidomics. Thirty-six SD rats were divided into a control group, a model group, SR-CR groups of different doses, and a simvastatin group, with six rats in each group. Rats in the control group were fed on a normal diet, while those in the remaining groups were fed on a high-lipid diet. After four weeks of feeding, drug treatment was carried out and rats were sacrificed after 12 weeks. Serum liver function and lipid indexes were detected using kits, and the pathomorphology of liver tissues was evaluated by hematoxylin-eosin(HE) staining and oil red O staining. Changes in lipid levels in rats were detected using the LC-MS technique. Differential lipid metabolites were screened by multivariate statistical analysis, and lipid metabolic pathways were plotted. The changes in lipid-related protein levels were further verified by Western blot. The results showed that compared with the control group, the model group showed increased levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P<0.01), and decreased levels of γ-glutamyl transferase(γ-GT) and high-density lipoprotein cholesterol(HDL-c)(P<0.01), which were significantly recovered by the intervention of SR-CR. HE staining and oil red O staining showed that different doses of SR-CR could reverse the steatosis in the rat liver in a dose-dependent manner. After lipidomics analysis, there were significant differences in lipid metabolism between the model group and the control group, with 54 lipids significantly altered, mainly including glycerolipids, phosphatidylcholine, and sphingolipids. After administration, 44 differential lipids tended to normal levels, which indicated that SR-CR groups of different doses significantly improved the lipid metabolism level in NAFLD rats. Western blot showed that SR-CR significantly decreased TG-synthesis enzyme 1(DGAT1), recombinant lipin 1(LPIN1), fatty acid synthase(FASN), acetyl-CoA carboxylase 1(ACC1), and increased the phosphorylation level of ACC1. These changes significantly decreased the synthesis of TG and increased the rate of its decomposition, which enhanced the level of lipid metabolism in the body and finally achieved the lipid-lowering effect. SR-CR can improve NAFLD by inhibiting the synthesis of fatty acids and TG.

Intermediate-length CGG repeat expansion in NOTCH2NLC is associated with pathologically confirmed Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Pathologically, it is characterized by ß-amyloid plaques and neurofibrillary tangles. There are several genes have been found to relate to AD, including the human-specific Notch2 N terminal-like C (NOTCH2NLC) gene. The CGG repeat expansion in NOTCH2NLC has been reported in clinically diagnosed AD patients. However, it has not been reported in pathologically confirmed AD cases. In this study, we detected the NOTCH2NLC CGG repeat expansion in pathologically confirmed AD brain samples by repeat-primed PCR (RP-PCR) and fluorescence amplicon length analysis PCR (AL-PCR). As a result, the intermediate-length CGG repeat expansion in NOTCH2NLC was validated in one out of 39 pathologically confirmed AD cases. Pathologically, p62 positive intranuclear inclusions were observed in wide brain areas, and most inclusions appeared to be presented in the glial cells. In summary, our study found that the intermediate-length CGG repeat expansion in NOTCH2NLC was associated with pathologically confirmed AD. The p62-positive intranuclear inclusions could co-exist with AD neuropathologic changes. These data suggest that the association of NOTCH2NLC CGG repeat expansion with AD may be stronger than in previous studies.

Significant Overlap of α-Synuclein, Amyloid-ß, and Phospho-Tau Pathologies in Neuropathological Diagnosis of Lewy-related Pathology: Evidence from China Human Brain Bank.

BACKGROUND: Lewy-related pathology (LRP), primarily comprised of α-synuclein, is a typical neuropathological change that has been identified in many neurodegenerative disorders such as Parkinson's disease (PD), PD with dementia, and dementia with Lewy bodies. OBJECTIVE: To investigate the distribution of LRP in the China Human Brain Bank, the co-occurrence of neuropathologic features of Alzheimer's disease (AD) in LRP cases, and LRP-related cognitive dysfunction. METHODS: LRP neuropathological diagnosis was performed in 180 postmortem brains. AD neuropathological diagnosis was then performed in the 21 neuropathologically-diagnosed LRP cases. Antemortem cognitive functioning evaluation (Everyday Cognitive, ECog) was assessed for brain donors by the immediate kin of the donor within 24 hours after death. RESULTS: 12% (21 in 180) postmortem brains were neuropathologically diagnosed as LRP cases. 86% (18 in 21) aged above 80, 81% (17 in 21) LRP cases combined with AD neuropathology, and 62% (13 in 21) combined with both the intermediate or high-level amyloid-ß and phospho-tau pathologies. ECog scores showed significant differences between the groups of LRP brainstem-predominant type and LRP diffuse neocortical type, and between groups of AD and the combined LRP (diffuse neocortical type)-AD. CONCLUSION: The overlap of neocortical α-synuclein, amyloid-ß, phospho-tau, and neuritic plaques in LRP suggested the potential interplay among the common characteristics of proteinopathies in the late stage of neuropathological development of LRP in human brains. The anatomic progression of LRP, the process of α-synuclein spreading from the brainstem to limbic and neocortical regions, might aggravate the deterioration of cognitive function in addition to that effect of AD.

Contribution of Alzheimer's disease neuropathologic change to the cognitive dysfunction in human brains with Lewy body-related pathology.

This study investigated the clinicopathological relationship between cognitive dysfunction and Lewy body-related pathology (LRP), and the role of Alzheimer's disease neuropathologic change (ADNC) in affecting this relationship in the Chinese population. A total of 127 brains with antemortem cognition assessment were collected. The postmortem neuropathological classification of LRP and staging of ADNC were evaluated. Pairwise correlation and ordered logistic regression analysis showed that LRP had a moderate correlation with Global Everyday Cognition scores. The proportion of the people with intermediate and high levels of comorbid ADNC increased with the deterioration of LRP. The fit of the cognition prediction model improved when we incorporated both LRP and ADNC into the model compared with LRP alone. Our study indicated that comorbid ADNC can variably present in patients with Lewy body disease. A combination of LRP and concurrent ADNC improves the prediction of cognitive dysfunction compared with LRP alone. These findings may suggest the potential benefit of combined therapeutic approaches targeting concurrent pathological pathways for the Lewy body diseases in the Chinese population.

Fabrication of Hydrophobic Ni Surface by Chemical Etching.

Hydrophobic surfaces were successfully fabricated on pure nickel substrates by a one-step chemical etching process with different acidic solutions. The static water contact angle (SCA) of the etched Ni surfaces reached higher than 125°, showing excellent hydrophobicity. The examination of surface chemical compositions implied that there were almost no polar moieties on the surface after chemical etching, except part of the surface was oxidized. After chemical etching, the nickel surfaces became much rough with packed terrace-/crater-/thorn-like clusters. According to the analysis of surface composition and morphology, the hydrophobicity was evidently attributed to the rough microstructures on the etched Ni surface. The best hydrophobicity on Ni surface was produced with the SCAs as high as 140.0° by optimizing the etching time and etchants. The results demonstrate that it is possible to construct hydrophobic surfaces on hydrophilic substrates by tailoring the surface microstructure using a simple chemical etching process without any further hydrophobic modifications by low surface energy materials.

Correlations Between Single Nucleotide Polymorphisms, Cognitive Dysfunction, and Postmortem Brain Pathology in Alzheimer's Disease Among Han Chinese.

In this study, the distribution of five Alzheimer's disease (AD)-related single nucleotide polymorphisms (SNPs) in the Han population was examined in combination with the evaluation of clinical cognition and brain pathological analysis. The associations among SNPs, clinical daily cognitive states, and postmortem neuropathological changes were analyzed in 110 human brains from the Chinese Academy of Medical Sciences/Peking Union Medical College (CAMS/PUMC) Human Brain Bank. APOE ε4 (OR = 4.482, P = 0.004), the RS2305421 GG genotype (adjusted OR = 4.397, P = 0.015), and the RS10498633 GT genotype (adjusted OR = 2.375, P = 0.028) were associated with a higher score on the ABC (Aß plaque score, Braak NFT stage, and CERAD neuritic plaque score) dementia scale. These results advance our understanding of the pathogenesis of AD, the relationship between pathological diagnosis and clinical diagnosis, and the SNPs in the Han population for future research.

Analysis of Brain Donors' Demographic and Medical Characteristics to Facilitate the Construction of a Human Brain Bank in China.

The Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS/PUMC) Human Brain Bank was established in December 2012 and had accomplished 197 brain donations by November 2017. The brain bank was based on a large-scale willed body donation program in CAMS/PUMC starting from 1999. Demographic and medical characteristic analysis of brain donors was conducted to facilitate the construction of the brain bank. The average postmortem delay of brain donors was 17.7 h and 77.7% of these donors died less than 15 km away from the brain bank. Donors were predominantly with higher-level education (p < 0.001) and at an older age when registration (p < 0.001) and donation (p < 0.001) occurred. Our results elucidated the characteristics of donors in the CAMS/PUMC Human Brain Bank, which may provide useful information to target potential donors and improve the quality and quantity of brain specimens. The current study may pave the way for the construction of a nationwide network of standardized human brain banks in China.

Quantitative protein profiling of hippocampus during human aging.

The hippocampus appears commonly affected by aging and various neurologic disorders in humans, whereas little is known about age-related change in overall protein expression in this brain structure. Using the 4-plex tandem mass tag labeling, we carried out a quantitative proteomic study of the hippocampus during normal aging using postmortem brains from Chinese subjects. Hippocampal samples from 16 subjects died of non-neurological/psychiatric diseases were divided into 4 age groups: 22-49, 50-69, 70-89, and >90. Among 4582 proteins analyzed, 35 proteins were significantly elevated, whereas 25 proteins were downregulated, along with increasing age. Several upregulated proteins, including transgelin, vimentin, myosin regulatory light polypeptide 9, and calcyphosin, were further verified by quantitative Western blot analysis of hippocampal tissues from additional normal subjects. Bioinformatic analysis showed that the upregulated and downregulated proteins were largely involved in several important protein-protein interaction networks. Proteins in the electron transport chain and synaptic vesicle fusion pathway were consistently downregulated with aging, whereas proteins associated with Alzheimer's disease showed little change. Our study demonstrates substantial protein profile changes in the human hippocampus during aging, which could be of relevance to age-related loss of hippocampal functions.

Actin binding proteins, spermatid transport and spermiation.

The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

Interaction of oligomeric breast cancer resistant protein (BCRP) with adjudin: a male contraceptive with anti-cancer activity.

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.