Age-related genes USP2 and ARG2 are involved in the reduction of immune cell infiltration in elderly patients with rheumatoid arthritis.

BACKGROUND: There are large differences in clinical manifestations and biological markers between elderly patients with rheumatoid arthritis (EPRA, age >60) and younger patients with RA (YPRA, age ≤60), partly owing to variations in the immune system of different age groups. Here, we focused on the changes of immune cell infiltration in YPRA and EPRA. METHODS: The R packages "ssGSEA" and "GSEA" were used to identify the changes in immune cell infiltration and immune-related pathways between the two groups. The R packages "WGCNA" and "DEseq2" were used to screen and verify age-related differentially expressed genes (DEGs). Hub genes were identified using Cytoscape and cytoHubba. Spearman correlation coefficient was conducted to evaluate correlations between hub age-related genes and immune cells. RESULTS: Compared with 54 established YPRA, several immune cells and immune-related pathways were markedly decreased in 29 EPRA synovial tissues. Moreover, 78 age-related DEGs related to amino acid and glycosphingolipid synthesis and metabolism were identified. USP2 and ARG2 were verified to be upregulated in EPRA, signifying that these two genes could effectively distinguish YPRA and EPRA and have potential as biomarkers. In addition, we found that USP2 was significantly negatively correlated with B cells and monocytes, while there was a significant negative association between ARG2 and T cells. CONCLUSIONS: In conclusion, this study is the first to systematically analyze changes in immune cell infiltration between YPRA and EPRA patients and obtain hub age-related genes, which may provide the basis for illuminating the pathogenesis of EPRA and informing treatment strategies.

Endolymphatic sac tumor misdiagnosed as metastatic renal cell carcinoma: Pitfalls in morphology and immunohistochemistry.

Endolymphatic sac tumor (ELST) is a rare disease that originates from the endolymphatic sac system of the inner ear. Being a low-grade malignant tumor, ELST has a mild morphology and is characterized by a slow but aggressive growth. Most clinicians and pathologists are unfamiliar with this disease. ELST can be misdiagnosed as metastatic renal cancer because of the similarity in morphology and expression of nephrogenic markers such as PAX8. The presented case of a 27-year-old man revealed that observing the characteristic location and confirming the absence of renal neoplasm to rule out the possibility of metastasis are critical for obtaining an accurate final diagnosis.

Interstitial lung disease in adult patients with anti-NXP2 antibody positivity: a multicentre 18-month follow-up study.

OBJECTIVES: Anti-nuclear matrix protein 2 (NXP2) antibody is a rare myositis-specific antibody. Thus, the pattern and prognosis of interstitial lung disease (ILD) in NXP2-positive patients remain unclear. This study investigates the clinical features and effects of pulmonary complications on survival in NXP2-positive patients. METHODS: We retrospectively analysed the clinical and follow-up data of a cohort of 33 hospitalised adult patients with anti-NXP2 antibody positivity at three tertiary rheumatology centres from June 2017 to December 2020. RESULTS: Thirty-three patients were enrolled, and 87.9% (29/33) had dermatomyositis. The major pulmonary lesions manifested as various types of ILD (14/33, 42.4%), bilateral pleural effusion (2/33, 6.1%) and diffuse alveolar haemorrhage (1/33, 3%). Only 3 patients (3/33, 9.1%) had respiratory symptoms at onset. The most common lung imaging manifestations were non-specific interstitial pneumonia (NSIP) and/or organising pneumonia (OP) (11/14, 78.6%). Patients in the ILD group were older than those in the non-ILD group (p=0.002). Logistic regression analysis showed that age (p=0.008) was the only independent predictor for ILD. Kaplan-Meier survival curves displayed no association between ILD and all-cause death (log-rank p=0.84). None of the deaths during follow-up were directly related to ILD. CONCLUSIONS: Adult patients with anti-NXP2 antibody positivity mainly had dermatomyositis. Concurrent ILD is not uncommon, but clinical manifestations are often latent. NSIP and/or OP are the most common patterns. ILD is more common in older age groups. Although the prognosis of patients in the ILD group is not very poor, early screening may help to improve prognosis and quality of life.

Validity and reliability of upper extremity star excursion balance test in adolescent swimmers.

Background: Upper limb balance is one of the important physical fitness parameters for all populations, especially overhead athletes like swimmers. Upper extremity star excursion balance test (UESEBT) is a comprehensive dynamic balance assessment, this study aims to explore the reliability and validity of UESEBT among adolescent swimmers. Methods: This cross-sectional study recruited 70 adolescent swimmers. All participants were required to complete UESEBT, upper quarter Y-balance test (UQYBT), maximal isometric strength (MIS) tests in upper limb, closed kinetic chain upper extremity stability test (CKCUEST), trunk flexor endurance test (TFET) and lateral trunk endurance test (LTET). The intra- and inter-operator reliability and the correlation of UESEBT with other physical performances were conducted. Results: For reliability, the intra- and inter-operator reliability of all directions and composite score were high-to-excellent (ICC = 0.706-1.000) among all participants. For validity, the UESEBT has a moderate-to-strong correlation with UQYBT (r = 0.42-0.72, p < 0.001), and a weak-to moderate one with CKCUEST (r = 0.25-0.42, p < 0.05). Furthermore, the UESEBT performance showed weak-to-moderate correlations with MIS (r = 0.24-0.44, p < 0.05). UESEBT was correlated to LTET (r = 0.24-0.33, p < 0.05) whereas no relationship was found with TFET. Conclusions: UESEBT was a reliable and valid tool to screen upper extremity dynamic balance among adolescent swimmers. UESEBT provides more detailed information in eight directions to assess the upper limb sport performance. Further study should explore the prediction ability of UESEBT for injury.

A non-small cell lung carcinoma patient responded to crizotinib therapy after alectinib-induced interstitial lung disease. / é˜¿æ¥æ›¿å°¼æ²»ç–—éžå°ç»†èƒžè‚ºç™Œæ‚£è€ å‘ç”Ÿé—´è´¨æ€§è‚ºç–¾ç— å†ç”¨å ‹å”‘æ›¿å°¼æ²»ç–—æœ‰æ•ˆ.

A 54-year-old, non-smoking woman was diagnosed as stage ⅣB adenocarcinoma with widespread bone metastasis (cT4N2M1c) in the First Affiliated Hospital, Zhejiang University School of Medicine. Immunohistochemistry result showed the presence of anaplastic lymphoma kinase (ALK) gene rearrangement; next-generation sequencing (NGS) indicated EML4-ALK fusion (E6:A20) with concurrent CCDC148-ALK (C1:A20), PKDCC-ALK (Pintergenic:A20)and VIT-ALK (V15:A20) fusions. After 32 weeks of alectinib treatment, the patient complained cough and exertional chest distress but had no sign of infection. Computed tomography (CT) showed bilateral diffuse ground glass opacities, suggesting a diagnosis of alectinib-related interstitial lung disease (ILD). Following corticosteroid treatment and discontinuation of alectinib, clinical presentations and CT scan gradually improved, but the primary lung lesions enlarged during the regular follow-up. The administration of crizotinib was then initiated and the disease was stable for 25 months without recurrence of primary lung lesions and ILD.

Locating Hydrogen Positions for COF-300 by Cryo-3D Electron Diffraction.

Covalent organic frameworks (COFs) have wide-ranging applications, and their host-guest interactions play an essential role in the achievement of COF functions. To investigate these host-guest interactions, it is necessary to locate all atoms, especially hydrogen atoms. However, it is difficult to determine the hydrogen atomic positions in COFs because of the complexities in synthesizing high-quality large single crystals. Three-dimensional electron diffraction (3D ED) has unique advantages for the structural determination of nanocrystals and identification of light atoms. In this study, it was demonstrated for the first time that the hydrogen atoms of a COF, not only on the framework but also on the guest molecule, can be located by 3D ED using continuous precession electron diffraction tomography (cPEDT) under cryogenic conditions. The host-guest interactions were clarified with the location of the hydrogen atoms. These findings provide novel insights into the investigation of COFs.

Stabilization of Extra-Large-Pore Zeolite by Boron Substitution for the Production of Commercially Applicable Catalysts.

Stable extra-large-pore zeolites are desirable for industrial purposes due to their ability to accommodate bulky reactants and diffusion through channels. Although there are several extra-large pore zeolites reported, stable ones are rare. Thus, their stabilization is a feasible strategy for industrial applications. Here, an extra-large-pore zeolite EWT with boron substitution is presented, and the resulting zeolite B-RZM-3 increased the thermal stability from 600 °C in its silica form to 850 °C. The crystal structure, determined by combining continuous rotation electron diffraction (cRED) and powder X-ray diffraction (PXRD), shows that B atoms preferentially substitute the interrupted (HO)T(OT)3 (Q3 ) sites and are partially converted into 3-coordination to relax framework deformation upon heating. After Al-reinsertion post-treatment, Al-B-RZM-3 shows higher ethylbenzene selectivity and ethylene conversion rate per mol acid site than commercial ZSM-5 and Beta zeolite in benzene alkylation reaction. Synthesizing extra-large-pore zeolite in borosilicate form is a potential approach to stabilize interrupted zeolites for commercial applications.

Recombinant VLPs empower RBM peptides showing no immunogenicity in native SARS-COV-2 protein to elicit a robust neutralizing antibody response.

New SARS-COV-2 vaccine strategies are still urgently needed, especially for emerging virus mutations and variants. In this study, we focused on analyzing the antigenicity and vaccine potency of linear peptide epitopes located in receptor binding motif (RBM) of spike (S) protein. Nine 12 to 16-mer overlapping peptides (P1-P9) were synthesized chemically and coupled to carrier protein KLH for the immunization in mice. Four of identified peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. Antisera obtained from VLPs -immunized mice demonstrated strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro. This study indicates that recombinant VLPs empower peptides which display underprivileged antigenicity in native protein to elicit high levels of neutralizing antibody, providing potential epitope candidates and an effective delivery strategy for the development of a multi-epitope vaccine.

RBD-Modified Bacterial Vesicles Elicited Potential Protective Immunity against SARS-CoV-2.

The disease caused by SARS-CoV-2 infection threatens human health. In this study, we used high-pressure homogenization technology not only to efficiently drive the bacterial membrane to produce artificial vesicles but also to force the fusion protein ClyA-receptor binding domain (RBD) to pass through gaps in the bacterial membrane to increase the contact between ClyA-RBD and the membrane. Therefore, the load of ClyA-RBD on the membrane is substantially increased. Using this technology, we constructed a "ring-like" bacterial biomimetic vesicle (BBV) loaded with polymerized RBD (RBD-BBV). RBD-BBVs injected subcutaneously can accumulate in lymph nodes, promote antigen uptake and processing, and elicit SARS-CoV-2-specific humoral and cellular immune responses in mice. In conclusion, we evaluated the potential of this novel bacterial vesicle as a vaccine delivery system and provided a new idea for the development of SARS-CoV-2 vaccines.

Guest-Binding-Induced Interhetero Hosts Charge Transfer Crystallization: Selective Coloration of Commonly Used Organic Solvents.

Unprecedented interheteromacrocyclic hosts charge transfer (CT) crystals were generated by cooling organic solutions containing p-dimethoxybenzene-constituted pillar[5]arene (P5A) and p-benzoquinone-constituted pillar[5]quinone (P5Q). Despite the weak CT interaction known between p-dimethoxybenzene and p-benzoquinone and the lack of formation of CT complexes between P5A and P5Q in the solution phase, CT cocrystals between P5A and P5Q were formed with solvent molecules included into the hosts' cavities. Such a cocrystallization arises from an elegant synergy between the CT interaction and solvent-binding-promoted crystallization. The interhetero hosts CT crystals were studied by optical and electron microscopic techniques, X-ray powder diffraction, solid-state NMR, UV-vis, IR spectroscopic studies, and X-ray single-crystal studies. The solvent complexation was critical for formation of the supramolecular CT microcrystals. The CT absorption bands faded upon removing the solvent molecules under vacuum, but they could be recovered by reuptake of the solvent molecules. Intriguingly, the CT absorption bands and uptake kinetics are distinguishably different for various organic solvents, thus providing a unique way to distinguish between different commonly used chemicals.

Blood tumor mutation burden can predict the clinical response to immune checkpoint inhibitors in advanced non-small cell lung cancer patients.

BACKGROUND: Tissue tumor mutation burden (tTMB) assessed by whole-exome sequencing (WES), which has been regarded as the gold standard method of tTMB measurement, can predict the clinical benefits of immune checkpoint inhibitors (ICIs). Multiple studies have investigated the feasibility of utilizing large panels to evaluate TMB but have obtained conflicting results. Furthermore, whether blood TMB (bTMB) can also be a predictive biomarker in NSCLC has not been determined. METHODS: Fifty-six advanced NSCLC patients treated with ICIs were enrolled, including an exploratory cohort (n = 42) and a small independent validation cohort (n = 14). Next-generation sequencing was performed on tumor and plasma samples collected prior to ICI treatment using a panel consisting of 520 cancer-related genes (OncoScreen) to evaluate tTMB/bTMB. WES was also performed on tumor samples to serve as references. RESULTS: A positive correlation between tTMB derived from WES and OncoScreen was observed. OncoScreen-derived tTMB showed a positive correlation with OncoScreen-derived bTMB. Patients with OncoScreen-derived tTMB [Formula: see text] 7 mutations/Mb (p = 0.003) or bTMB [Formula: see text] 11 mutations/Mb (p = 0.0029) had superior progression-free survival (PFS). In the small validation cohort, patients with OncoScreen-derived bTMB [Formula: see text] 11 mutations/Mb exhibited longer PFS (p = 0.192) with a nonsignificant difference. In all 42 patients who had available bTMB and PFS, patients with bTMB [Formula: see text] 11 mutations/Mb had significantly longer PFS (p = 0.011) than those with bTMB [Formula: see text] 11 mutations/Mb. CONCLUSION: Our study confirmed the feasibility of using large panels to estimate TMB. We also demonstrated that bTMB can serve as a potential biomarker for predicting the efficacy of ICIs in NSCLC.

Forced vital capacity predicts the survival of interstitial lung disease in anti-MDA5 positive dermatomyositis: a multi-centre cohort study.

OBJECTIVES: Anti-melanoma differentiation-associated gene 5 (MDA5) positive DM is a life-threatening disease often complicated with rapidly progressive interstitial lung disease (ILD). This study aimed to establish and validate a clinical prediction model for 6-month all-cause mortality in Chinese patients with anti-MDA5 positive DM-ILD. METHODS: We conducted a retrospective observational study using a single-centre derivation cohort and a multicentre validation cohort. Hospitalized DM patients with positive anti-MDA5 antibody and ILD course ≤3 months on admission were included. Patients' baseline characteristics were described and compared between the deceased and survivors by univariable Cox regression. Optimal cut-off values were defined by the 'survminer' R package for significant continuous variables. Independent prognostic factors were determined by the final multivariable Cox regression model chosen by backward stepwise algorithm, which could be reproduced in both cohorts. The Kaplan-Meier survival analyses based on the derived predictor were conducted. RESULTS: A total of 184 and 81 eligible patients were included with a cumulative 40.8 and 40.7% 6-month mortality in the derivation and validation cohorts, respectively. Based on multivariable Cox regression, the prognostic factor at baseline was identified and validated as three-category forced vital capacity (FVC)%: FVC% ≥50%, FVC% <50%, unable to perform. This significantly distinguishes three risk stages with mortalities of 15.3, 46.8, 97.4% in the derivation cohort, and 14.9, 58.3, 86.4 in the validation cohort, respectively (all P <0.05). CONCLUSION: The validated FVC%-based categorical predictor in anti-MDA5 positive DM-ILD is helpful for risk stratification in clinical practice and might facilitate cohort enrichment for future trials.

Zn(II)-curcumin solid dispersion impairs hepatocellular carcinoma growth and enhances chemotherapy by modulating gut microbiota-mediated zinc homeostasis.

Zinc(II) complexes of curcumin display moderate cytotoxicity towards cancer cells at low micromolar concentrations. However, the clinical use of zinc(II) complexes is hampered by hydrolytic insolubility and poor bioavailability and their anticancer mechanisms remain unclear. Here, we investigated the efficacy and mechanism of action of a polyvinylpyrrolidone (PVP-k30)-based solid dispersion of Zn(II)-curcumin (ZnCM-SD) against hepatocellular carcinoma (HCC) in vitro and in vivo. In vitro assays revealed ZnCM-SD not only reduced the viability of HepG2 cells and SK-HEP1 cells in a dose-dependent manner, but also potently and synergistically enhanced cell growth inhibition and cell death in response to doxorubicin by regulating cellular zinc homeostasis. ZnCM-SD was internalized into the cells via non-specific endocytosis and degraded to release curcumin and Zn2+ ions within cells. The anticancer effects also occur in vivo in animals following the oral administration of ZnCM-SD, without significantly affecting the weight of the animals. Interestingly, ZnCM-SD did not reduce tumor growth or affect zinc homeostasis in HepG2-bearing mice after gut microbiome depletion. Moreover, administration of ZnCM-SD alone or in combination with doxorubicin significantly attenuated gut dysbiosis and zinc dyshomeostasis in a rat HCC model. Notably, fecal microbiota transplantation revealed the ability of ZnCM-SD to regulate zinc homeostasis and act as a chemosensitizer for doxorubicin were dependent on the gut microbiota. The crucial role of the gut microbiota in the chemosensitizing ability of ZnCM-SD was confirmed by broad-spectrum antibiotic treatment. Collectively, ZnCM-SD could represent a simple, well-tolerated, safe, effective therapy and function as a novel chemosensitizing agent for cancer.

A novel compound heterozygous variant identified in GLDC gene in a Chinese family with non-ketotic hyperglycinemia.

BACKGROUND: Non-ketotic hyperglycinemia (NKH) is a rare, devastating autosomal recessive disorder of glycine metabolism with a very poor prognosis. Currently, few studies have reported genetic profiling of Chinese NKH patients. This study aimed to identify the genetic mutations in a Chinese family with NKH. METHODS: A Chinese family of Han ethnicity, with three siblings with NKH was studied. Sanger sequencing and multiplex ligation-dependent probe amplification combined with SYBR green real-time quantitative PCR was used to identify potential mutations in the GLDC, AMT and GCSH genes. The potential pathogenicity of the identified missense mutation was analyzed using SIFT, PolyPhen-2, PROVEAN and MutationTaster software. RESULTS: All patients exhibited severe and progressive clinical symptoms, including lethargy, hypotonia and seizures, and had greatly elevated glycine levels in their plasma and CSF. Molecular genetic analysis identified compound heterozygous variants in the GLDC gene in these three siblings, including a novel missense variant c.2680A > G (p.Thr894Ala) in exon 23 and a heterozygous deletion of exon 3, which were inherited respectively from their parents. In silico analysis, using several different types of bioinformatic software, predicted that the novel variant c.2680A > G in the GLDC gene was pathogenic. Moreover, the deletion of exon 3 was identified for the first time in a Chinese population. CONCLUSIONS: A novel missense variant and a previously reported deletion in GLDC gene were identified. The two variants of GLDC gene identified probably underlie the pathogenesis of non-ketotic hyperglycinemia in this family, and also enrich the mutational spectrum of GLDC gene.

Comparison of Moderate and High Energy of a Nano-Fractional Radiofrequency Treatment on a Photoaging Hairless Mice Model.

BACKGROUND: Fractional radiofrequency (FRF) has been widely used in skin rejuvenation. To explore optimal settings, it is important to compare different treatment parameters. OBJECTIVE: This study was designed to compare the effect of moderate-energy and high-energy FRF treatment on a hairless mice model. METHODS: Fifteen photoaged hairless mice were assigned to 3 groups: control, moderate energy, and high energy. Two treatment sessions (T × 1 and T × 2) were performed at 1-month interval. Transepidermal water loss was measured at baseline, immediately, 1, 2, and 4 weeks after T × 1. Skin samples were harvested before each treatment, 1 and 2 months after T × 2. Neocollagenesis was evaluated by hematoxylin and eosin staining, Masson staining, and immunohistochemistry analysis. RESULTS: Transepidermal water loss of high-energy group was significantly higher than the moderate-energy group (p = .008) immediately after T × 1. Remarkable fibroblast proliferation was observed at 1 month after T × 1, followed by significant dermal thickening, and increase of Type I collagen and Type III collagen. There was no significant difference between 2 energy groups in fibroblast proliferation, dermal thickness, and collagen density. CONCLUSION: The effect of moderate-energy treatment was comparable with that of high energy in neocollagenesis, whereas moderate energy yielded less damage to skin barrier function.

Objective evaluation of the effects of intense pulsed light treatment on Asian skin by reflectance confocal microscopy analysis.

This study aimed to evaluate the effect of IPL treatment on Asian skin by reflectance confocal microscopy (RCM) analysis. Ten Asian female volunteers (39~54 years old, Fitzpatrick skin type III~IV) received five monthly IPL treatments. RCM skin images were evaluated, and several skin physiological parameters including thickness of stratum corneum, minimal thickness of epidermis, thickness of basal layer, density of dermal papillae, and mean diameter of papillae capillaries were measured both at baseline and 1 month after the last treatment. Transepidermal water loss (TEWL) was evaluated, as well. Thickness of stratum corneum was 4.80 ± 1.48 µm before IPL treatment and 5.50 ± 1.35 µm after treatment (p = 0.322). Both minimal thickness of epidermis and thickness of basal layer were significantly increased (p = 0.002 and 0.018, respectively) after IPL treatment. Dermal papillae density was significantly increased (p = 0.035), whereas mean capillary diameter was reduced significantly (p = 0.035). TEWL was slightly increased after treatment, while the difference was not significant on either T-zone or U-zone (p = 0.085 on T-zone and p = 0.114 on U-zone). RCM imaging is a feasible method to evaluate the effect of IPL effect on human skin. Moreover, IPL treatment serves to be highly safe in skin rejuvenation.

[Expression of leukocyte immunoglobulin-like receptor A3 in CD14+ monocytes of patients with rheumatoid arthritis].

OBJECTIVE: To investigate the expression of leukocyte immunoglobulin-like receptor A3 (LILRA3) in CD14+ monocytes of patients with rheumatoid arthritis (RA). METHODS: Fifty three RA patients admitted in the Second Affiliated Hospital of Zhejiang University School of Medicine from February 2017 to August 2017, and 21 healthy subjects were enrolled in the study. The expression of LILRA3 in CD14+ monocyte subset was determined by flow cytometry, and its correlations with clinical features, laboratory examination results, antibodies and disease activity were analyzed. RESULTS: LILRA3 percentage in the CD14+ monocyte subset of RA patients was higher than that in the healthy controls (P<0.01). The percentage of LILRA3 was positively correlated with number of tenderness joints, number of swollen joints and erythrocyte sedimentation rate (r=0.280, 0.371, 0.341, P <0.05 or <0.01), but was not correlated with the age, course of disease, Sharp score, C reactive protein, blood routine index and immunoglobulin (all P>0.05). In addition, the percentages of LILRA3 in the monocytes of rheumatoid factor (RF)-positive or anti-cyclic citrullinated peptide (CCP) antibody-positive patients were significantly higher than those of the RF-or anti-CCP antibody-negative patients (all P < 0.05); and the percentage of LILRA3 in patients with DAS28>5.1 was higher than that in patients with DAS28 ≤ 5.1 (P<0.05). CONCLUSIONS: The expression of LILRA3 is up-regulated in CD14+ monocyte subset isolated from RA patients, and it is correlated with disease activity.

Genome-wide alteration of 5-hydroxymenthylcytosine in a mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of neurodegenerative disorder that leads to a decline in cognitive function. In AD, aggregates of amyloid ß peptide precede the accumulation of neurofibrillary tangles, both of which are hallmarks of the disease. The great majority (>90 %) of the AD cases are not originated from genetic defects, therefore supporting the central roles of epigenetic modifications that are acquired progressively during the life span. Strong evidences have indicated the implication of epigenetic modifications, including histone modification and DNA methylation, in AD. Recent studies revealed that 5-hydroxymethylcytosine (5hmC) is dynamically regulated during neurodevelopment and aging. RESULTS: We show that amyloid peptide 1-42 (Aß1-42) could significantly reduce the overall level of 5hmC in vitro. We found that the level of 5hmC displayed differential response to the pathogenesis in different brain regions, including the cortex, cerebellum, and hippocampus of APP-PSEN1 double transgenic (DTg) mice. We observed a significant decrease of overall 5hmC in hippocampus, but not in cortex and cerebellum, as the DTg mice aged. Genome-wide profiling identified differential hydroxymethylation regions (DhMRs) in DTg mice, which are highly enriched in introns, exons and intergenic regions. Gene ontology analyses indicated that DhMR-associated genes are highly enriched in multiple signaling pathways involving neuronal development/differentiation and neuronal function/survival. CONCLUSIONS: 5hmC-mediated epigenetic regulation could potentially be involved in the pathogenesis of AD.