Characterization of Mitochondrial DNA Methylation of Alzheimer's Disease in Plasma Cell-Free DNA.

Noninvasive diagnosis of Alzheimer's disease (AD) is important for patients. Significant differences in the methylation of mitochondrial DNA (mtDNA) were found in AD brain tissue. Cell-free DNA (cfDNA) is a noninvasive and economical diagnostic tool. We aimed to characterize mtDNA methylation alterations in the plasma cfDNA of 31 AD patients and 26 age- and sex-matched cognitively normal control subjects. We found that the mtDNA methylation patterns differed between AD patients and control subjects. The mtDNA was predominantly hypomethylated in the plasma cfDNA of AD patients. The hypomethylation sites or regions were mainly located in mt-rRNA, mt-tRNA, and D-Loop regions. The hypomethylation of the D-Loop region in plasma cfDNA of AD patients was consistent with that in previous studies. This study presents evidence that hypomethylation in the non-protein coding region of mtDNA may contribute to the pathogenesis of AD and potential application for the diagnosis of AD.

Initial low-dose computed tomography screening results and summary of participant characteristics: based on the latest Chinese guideline.

Background: Low-dose computed tomography (LDCT) has been promoted as a promising screening strategy for early detection of lung cancer. China released the latest lung cancer screening guideline in 2021. The compliance of the individuals who received LDCT for lung cancer screening with the guideline is unknown yet. It is necessary to summarize the distribution of guideline-defined lung cancer-related risk factors in the Chinese population so as to inform the selection of target population for the future lung cancer screening. Methods: A single-center, cross-sectional study design was adopted. All participants were individuals who underwent LDCT at a tertiary teaching hospital in Hunan, China, between 1 January and 31 December 2021. LDCT results were derived along with guideline-based characteristics for descriptive analysis. Results: A total of 5,486 participants were included. Over one-quarter (1,426, 26.0%) of the participants who received screening did not meet the guideline-defined high-risk population, even among non-smokers (36.4%). Most of the participants (4,622, 84.3%) were found to have lung nodules, while no clinical intervention was required basically. The detection rate of positive nodules varied from 46.8% to 71.2% when using different cut-off values for positive nodules. Among non-smoking women, ground glass opacity appeared to be more significantly common compared with non-smoking men (26.7% vs. 21.8%). Conclusion: Over one-quarter of individuals who received LDCT screening did not meet the guideline-defined high-risk populations. Appropriate cut-off values for positive nodules need to be continuously explored. More precise and localized criteria for high-risk individuals are needed, especially for non-smoking women.

ADmeth: A Manually Curated Database for the Differential Methylation in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease. More and more evidence show that DNA methylation is closely related to the pathological mechanism of AD. Many AD-associated differentially methylated genes, regions and CpG sites have been identified in recent researches, which may have great potential in clinical research. However, there is no dedicated database to collect AD-related differential methylation up to now. To provide a reference to researchers, we design a database named ADmeth by manually curating relevant articles, which contains a total of 16,709 AD-related differentially methylated items identified from different brain regions and different cell types in the blood, involving 209 genes, 2,229 regions and 14,271 CpG sites. The ADmeth database provides user-friendly pages to search, submit and download data. We hope that the ADmeth database can facilitate researchers to select candidate AD-associated methylation markers in revealing the pathological mechanism of AD and promote the cell-free DNA based non-invasive diagnosis of AD. The ADmeth database is available at http://www.biobdlab.cn/ADmeth.

Toehold-Mediated Strand Displacement Reaction for Dual-Signal Electrochemical Assay of Apolipoprotein E Genotyping.

Apolipoprotein E (apoE) polymorphic genes are one of the main genetic determinants of Alzheimer's disease (AD) risk. Relying on the toehold-mediated strand displacement reaction (SDR), the dual-signal electrochemical assay of apoE genotyping with potential applications in the early diagnosis of AD has been achieved. The displacement of the surface-confined methylene blue- and ferrocene-capped detection probe-modified gold nanoparticles (AuNPs) by the complementary sequences (Tc 1 and Tc 2, fragment of allele ε4 at codon 112 and that of allele ε3 or ε4 at codon 158, respectively), triggered by the highly specific SDR, results in decreased voltammetric signals. In contrast, partial strand displacement caused by the single mismatched sequences (Tsm 1 and Tsm 2, fragment of allele ε2 or ε3 at codon 112 and that of allele ε2 at codon 158, respectively) produces larger voltammetric signals. The proposed method serves as a versatile platform for the discrimination of six apoE genotypes, including three homozygotes (ε2/2, ε3/3, and ε4/4) and three heterozygotes (ε2/3, ε2/4, and ε3/4), and for the quantification of apoE ε3/3 from genomic DNA extracts of AD patients.

Targeted Sequencing of Genomic Repeat Regions Detects Circulating Cell-free Echinococcus DNA.

BACKGROUND: Echinococcosis is a chronic zoonosis caused by tapeworms of the genus Echinococcus. Treatment of the disease is often expensive and complicated, sometimes requiring extensive surgery. Ultrasonographic imaging is currently the main technique for diagnosis, while immunological analysis provides additional information. Confirmation still needs pathological analysis. However, these diagnostic techniques generally detect infection in late stages of the disease. An accurate, early and non-invasive molecular diagnostic method is still unavailable. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the cell-free DNA (cfDNA) from plasma of echinococcosis patients and confirmed the presence of Echinococcus DNA. To improve detection sensitivity, we developed a method based on targeted next-generation sequencing of repeat regions. Simulation experiments demonstrate that the targeted sequencing is sensitive enough to detect as little as 0.1% of an Echinococcus genome in 1 mL of plasma. Results obtained using patient plasma shows that the Area Under the Curve (AUC) of the method is 0.862, with a detection sensitivity of 62.50% and specificity of 100%, corresponding to a Youden-index of 0.625. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that hydatid cysts release cfDNA fragments into patient plasma. Using the repeat region targeted sequencing method, highly specific detection of Echinococcus infection was achieved. This study paves a new avenue for potential non-invasive screening and diagnosis of echinococcosis.

Dual-Channel Surface Plasmon Resonance for Quantification of ApoE Gene and Genotype Discrimination in Unamplified Genomic DNA Extracts.

Identification of gene variation is of great importance for attaining information related to disease susceptibility. A highly sensitive and specific surface plasmon resonance (SPR) method for quantification of the apoE gene and genotype discrimination was demonstrated. The complementary sequences with the specific recognition sites of GCGC bases upon hybridization to the preimmobilized biotinylated probes could be cleaved by the restriction enzyme HhaI, while the existence of the single-base mismatch (GTGC) prevented the cleavage reaction. In both cases, the incorporation of streptavidin increased the sensitivity of the SPR assay, and the detection levels of 10 fM and 50 fM for the complementary and single-base mismatched sequences were attained, respectively. The sensing protocol is simple, label-free, and quantitative, thus avoiding the complicated polymerase chain reaction (PCR) amplification procedures. The proposed method serves as a viable means for facile and sensitive analyses of apoE genes in four unamplified genomic DNA extracts.

Atorvastatin protects against cerebral ischemia/reperfusion injury through anti-inflammatory and antioxidant effects.

In addition to its lipid-lowering effect, atorvastatin exerts anti-inflammatory and antioxidant effects as well. In this study, we hypothesized that atorvastatin could protect against cerebral ischemia/reperfusion injury. The middle cerebral artery ischemia/reperfusion model was established, and atorvastatin, 6.5 mg/kg, was administered by gavage. We found that, after cerebral ischemia/reperfusion injury, levels of the inflammation-related factors E-selectin and myeloperoxidase were upregulated, the oxidative stress-related marker malondialdehyde was increased, and superoxide dismutase activity was decreased in the ischemic cerebral cortex. Atorvastatin pretreatment significantly inhibited these changes. Our findings indicate that atorvastatin protects against cerebral ischemia/reperfusion injury through anti-inflammatory and antioxidant effects.

De novo design of self-assembled hexapeptides as ß-amyloid (Aß) peptide inhibitors.

The ability of peptides to construct specific secondary structures provides a useful function for biomaterial design that cannot be achieved with traditional organic molecules and polymers. Inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Existing peptide-based inhibitors are mainly derived from original amyloid sequences, which have very limited sequence diversity and activity. It is highly desirable to explore other peptide-based inhibitors that are not directly derived from amyloid sequences. Here, we develop a hybrid high-throughput computational method to efficiently screen and design hexapeptide inhibitors against amyloid-ß (Aß) aggregation and toxicity from the first principle. Computationally screened/designed inhibitors are then validated for their inhibition activity using biophysical experiments. We propose and demonstrate a proof-of-concept of the "like-interacts-like" design principle that the self-assembling peptides are able to interact strongly with conformationally similar motifs of Aß peptides and to competitively reduce Aß-Aß interactions, thus preventing Aß aggregation and Aß-induced toxicity. Such a de novo design can also be generally applicable to design new peptide inhibitors against other amyloid diseases, beyond traditional peptide inhibitors with homologous sequences to parent amyloid peptides.

The current situation on vascular cognitive impairment after ischemic stroke in Changsha.

The objectives of the study were to explore the prevalence and effects of vascular cognitive impairment (VCI) among ischemic stroke patients and to provide a basis for prevention and treatment strategies. A stratified cluster random sampling method was performed, and 689 ischemic stroke patients (over 40 years of age) were enrolled. All of the patients had received a neuropsychological assessment battery to assess cognitive function and self-designed questionnaires to collect relevant information. According to the cognitive status, the patients were divided into two groups, a case group and a control group. The caregivers of the patients were given a questionnaire concerning the awareness of and attitudes toward VCI. In this study, we determined that the prevalence of VCI was 41.8%. Aging, paraventricular white matter lesion (WML), macroangiopathy, high levels of alcohol, a lack of hobbies, and excessive sleep were risk factors for vascular cognitive impairment no dementia (VCIND). A high level of education, manual-work, low level of alcohol use, regular health checks, a vegetable-based diet, and more fruit and milk were protective factors for VCIND. Living alone, hyperlipidemia, transient ischemic attack, a family history of stroke, and brain atrophy were risk factors of vascular dementia (VD). A high educational level, a vegetable-based diet, and tea were protective factors for VD. The general public awareness of VCI was found to be insufficient, and there was a prejudice toward and lack of funding for the care of VCI patients. The prevalence of VCI is high in ischemic stroke patients, and there are different impact factors at different stages. Despite the high prevalence of VCI, the general public awareness is limited. Appropriate prevention measures should be developed to reduce the prevalence of VCI.

NADPH oxidase mediates the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage.

Oxygen free radicals and their reactive lipid peroxidation are known to be elements promoting ischemia-reperfusion damage. NADPH oxidase is a major factor in peroxide production. Excessive production of oxygen free radicals is considered as an important mechanism in the expression of matrix metalloproteinase (MMP)-9 and in damage to the blood-brain barrier (BBB). In this study, we evaluated changes in the expression of the NADPH oxidase catalytic subunit gp91(phox) and oxidase activity, as well as the involvement of NADPH oxidase catalysis in the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage. A middle cerebral artery occlusion (MCAO) model was established using male Sprague-Dawley (SD) rats. Brain tissue was isolated for triphenyltetrazolium chloride (TTC) staining, gp91(phox) mRNA quantitative PCR analysis, western blot analysis, NADPH oxidase activity determination (detection), and MMP-9 gelatin zymography analysis. In the MCAO rats, gp91(phox) and MMP-9 expression was upregulated in the ischemic hemisphere of the brain tissue after 90 minutes of MCAO with 22·5 hours of reperfusion. Inhibition of NADPH oxidase with apocynin reduced the increase in MMP-9. These results suggest that NADPH oxidase is a major precipitating factor for the expression of MMP-9 in the ischemic brain tissue.

Protective and antioxidant effect of Danshen polysaccharides on cerebral ischemia/reperfusion injury in rats.

It is well documented that oxidative stress has been implicated as one of the leading causes for brain damage induced by cerebral ischemia/reperfusion (I/R). In this study, we assessed the potential cerebraprotective and antioxidant effects of the polysaccharides (DSP) from the roots of Salvia miltiorrhiza against global cerebral I/R injury in an animal model established by blocking bilateral common carotid arteries (BCCA) for 30 min followed by reperfusion for 24 h. The rats were treated with their respective treatments for 10 days prior to the BCCA occlusion. After that, animals were sacrificed by decapitation, brain was removed, and various biochemical estimations, cerebral edema and assessment of cerebral infarct size were carried out. We found that pretreatment with DSP significantly decreased the neurological deficit scores, percentage of infarction and brain edema and the generation of mitochondrial reactive oxygen species (ROS). Moreover, DSP also increased mitochondria superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and reduced malondialdehyde (MDA) production in cerebral ischemia brain. In conclusion, these studies suggest that pretreatment with DSP provides significant protection against cerebral I/R injury in rats most probably by virtue of its antioxidant property.

Reliability, validity, and optimal cutoff score of the montreal cognitive assessment (changsha version) in ischemic cerebrovascular disease patients of hunan province, china.

BACKGROUND/AIMS: The goal of this study was to examine the reliability and validity of the Changsha version of the Montreal Cognitive Assessment (MoCA-CS) in ischemic cerebrovascular disease patients of Hunan Province, China, and to explore the optimal cutoff score for detecting vascular cognitive impairment-no dementia (VCI-ND) and vascular dementia (VD). METHODS: Three hundred and thirty-eight ischemic cerebrovascular disease patients (131 with normal cognition, 111 with VCI-ND, and 96 with VD) and 132 healthy controls were recruited. All participants accepted examination by the MoCA-CS, Mini-Mental State Examination (MMSE), and other related scales. A detailed neuropsychological battery was used for making a final cognitive diagnosis. SPSS 16.0 statistical software was used for reliability, validity examination, and optimal cutoff score detection. RESULTS: Cronbach's α of the MoCA-CS was 0.884, and test-retest and interrater reliability of the MoCA-CS were 0.966 and 0.926, respectively. MoCA-CS scores were highly correlated with MMSE scores (r = 0.867) and simplified intelligence quotients (r = 0.822). The results indicate that 1 point should be added for subjects with less than 6 years of education, and that the optimal cutoff score for detecting VCI-ND is 26/27 (sensitivity 96.1%, specificity 75.6%), whereas the optimal cutoff score for detecting VD is 16/17 (sensitivity 92.7%, specificity 96.3%). CONCLUSION: The MoCA-CS has good reliability and validity, and is a useful cognitive screening instrument for detecting VCI in the Chinese population.