Progress in the research of COVID-19 effects on Alzheimer's disease / 中国热带医学

@#Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than one-third of patients with COVID-19 experience neurological symptoms, including confusion, headaches, and decreased/disordered taste. Alzheimer's disease (AD) is a slowly progressive neurodegenerative disease and the most common type of dementia. Alzheimer's disease patients are at high risk and susceptible to infection with COVID-19, which may cause severe illness and even death. There appears to be an interaction between AD and COVID-19, and on the one hand, patients with COVID-19 seem to be more likely to develop AD. AD patients, on the other hand, may be more susceptible to severe COVID-19. Therefore, understanding the common link between COVID-19 and AD may help to develop treatment strategies. Risk factors common to AD and COVID-19 are aging, ApoE ε4 allele, β-amyloid (Aβ) deposition, angiotensin-converting enzyme (ACE), neuroinflammation, oxidative stress. Here, this article focuses on the relationship between COVID-19 and AD, explores common risk factors and potential pathogenesis, and provides help for early prevention, treatment and recovery.

HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state.

HBV covalently closed circular DNA (cccDNA) is the template for the transcription of HBV. HBV core protein (HBc) is a main component of the HBV cccDNA minichromosome. However, the function of HBc in cccDNA is not fully understood. In light of recent findings that HBV cccDNA may be regulated epigenetically, we analyzed the binding of HBc to cccDNA and the impact of HBc on cccDNA epigenetic profile in the liver biopsy samples of 22 patients with chronic Hepatitis B (CHB). We found that HBc binding to HBV cccDNA occurred preferentially at CpG island 2, an important region for the regulation of HBV transcription. Furthermore, the relative abundances of HBc binding to CpG island 2 were positively correlated with the ratios of relaxed circular DNA to cccDNA and the levels of serum HBV DNA in those patients. Interestingly, the relative abundances of HBc binding to CpG island 2 were associated with the binding of CREB binding protein (CBP) and with hypomethylation in CpG island 2 of HBV cccDNA minichromosomes. However, relatively higher amounts of HBc binding to CpG island 2 of cccDNA were accompanied by lower amounts of HDAC1 binding. Multivariate analysis revealed that the abundances of HBc binding to CpG island 2 of cccDNA and positive HBeAg were independent factors associated with the replication of HBV (p = 0.001 for both). Apparently, HBc is a positive regulator of HBV transcription and replication, maintaining the permissive epigenetic state in the critical region of the HBV cccDNA minichromosomes.

[Screening of ADAMs that can mediate the generation of soluble MHC I].

AIM: To screen ADAMs that can mediate the generation of soluble MHCI using HEK293 cells are study model. METHODS: ADAM8, ADAM10, ADAM15 or ADAM17 cDNA expressing vector s were separately constructed with pcDNA3. 1/V5 as vectors and were transfected into HEK293 cells. Hygromycin B was used to screen the cells that stably expressed ADAMs. After HEK293 cells were sell-surface labeled with biotin and cultured for another 4 h and soluble MHC I the was examined by SDS-PAGE, Western blot and enhanced chemiluminescence. RESULTS: HEK293 cells stably expressing ADAMs were successfully obtained and the over-expression of ADAM10 or ADAM17 increased the release of soluble MHC I. CONCLUSION: ADAM10 and ADAM17 have the potentials of mediating the generation of soluble MHC I.

Inhibition of beta-lactamase-mediated oxacillin resistance in Staphylococcus aureus by a deoxyribozyme.

AIM: To investigate the oxacillin susceptibility restoration of methicillin-resistant Staphylococcus aureus (MRSA) by targeting the signaling pathway of blaR1- blaZ with a DNAzyme. METHODS: A DNAzyme (named PS-DRz602) targeting blaR1 mRNA was designed and synthesized. After DRz602 was introduced into a MRSA strain WHO-2, the colony-forming units of WHO-2 on the Mueller-Hinton agar containing 6 mg/L oxacillin and the minimum inhibitory concentrations of oxacillin were determined. The inhibitory effects of DRz602 on the expressions of antibiotic- resistant gene blaR1 and its downstream gene blaZ were detected by real time RT-PCR. RESULTS: PS-DRz602 significantly decreased the transcription of blaR1 mRNA and led to the significant reduction of blaZ in a concentration-dependent manner. Consequently, the resistance of S aureus WHO-2 to the beta-lactam antibiotic oxacillin was significantly inhibited. CONCLUSION: Our results indicated that blocking the blaR1-blaZ signaling pathway via DNAzyme might provide a viable strategy for inhibiting the resistance of MRSA to beta-lactam antibiotics and that BlaR1 might be a potential target for pharmacological agents combating MRSA.

[Rapid detection of rifampin resistance of Mycobacterium tuberculosis using high-throughput pyrosequencing technique].

OBJECTIVE: To develop a pyrosequencing approach to rapid detection of rifampin resistance in Mycobacterium tuberculosis based on characterization of all possible mutations in the 81 bp core region. METHODS: Two pyrosequencing sequencing primers and 1 pair of PCR primers were chosen for pyrosequencing analysis. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from Mycobacterium tuberculosis H(37) Rv strains. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical Mycobacterium tuberculosis isolates with known antibiotic phenotypes. RESULTS: Rifampin resistance could be determined within 2 hours after PCR amplification and single-stranded template preparation by using only two pyrosequencing reactions. About 50 fg DNA/reaction was required in order to get sufficient PCR product for producing a long, clear and accurate pyrosequencing pattern. A total of 41 Mycobacterium tuberculosis isolates were tested and the results were concordant with those based on drug susceptibility testing and conventional DNA sequencing. CONCLUSION: The pyrosequencing technique is simple to perform and can be used as a rapid, high-throughput and efficient method for detecting rifampin resistance in Mycobacterium tuberculosis.

A microarray-based gastric carcinoma prewarning system.

AIM: To develop a microarray-based prewarning system consisting of gastric cancer chip, prewarning data and analysis software for early detection of gastric cancer and pre-cancerous lesions. METHODS: Two high-density chips with 8,464 human cDNA sites were used to primarily identify potential genes specific for normal gastric mucosa, pre-cancerous lesion and gastric cancer. The low-density chips, composed of selected genes associated with normal gastric mucosa, precancerous lesion and gastric cancer, were fabricated and used to screen 150 specimens including 60 specimens of gastric cancer, 60 of pre-cancerous tissues and 30 of normal gastric mucosa. CAD software was used to screen out the relevant genes and their critical threshold values of expression levels distinguishing normal mucosa from pre-cancerous lesion and cancer. All data were stored in a computer database to establish a prewarning data library for gastric cancer. Two potential markers brcaa1 and ndr1 were identified by Western blot and immunohistochemistry. RESULTS: A total of 412 genes associated with three stages of gastric cancer development were identified. There were 216 genes displaying higher expression in gastric cancer, 85 genes displaying higher expression in pre-cancerous lesion and 88 genes displaying higher expression in normal gastric mucosa. Also 15 genes associated with metastasis of gastric cancer and 8 genes associated with risk factors were screened out for target genes of diagnosis chip of early gastric cancer. The threshold values of 412 selected genes to distinguish gastric cancer, pre-cancerous lesion from normal gastric mucosa were defined as 6.01+/-2.40, 4.86+/-1.94 and 5.42+/-2.17, respectively. These selected 412 genes and critical threshold values were compiled into an analysis software, which can automatically provide reports by analyzing the results of 412 genes obtained by examining gastric tissues. All data were compiled into a prewarning database for gastric cancer by CGO software. Northern blot and immunohistochemistry analysis confirmed that gene and protein of brcaa1 displayed lower expression in normal gastric mucosa and higher expression in gastric cancer tissues, conversely, ndr1 displayed lower expression in gastric cancer and higher expression in normal gastric mucosa. CONCLUSION: The microarray-based prewarning system for gastric cancer was developed. This system consisted of gastric cancer-associated gene chip, prewarning data and analysis software, which has a high potential for applications in the early detection of gastric cancer. The two potential markers brcaa1 and ndr1 identified may be used to distinguish cancer status fand non-cancer status.

Pyrosequencing-based approach for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Rifampin resistance in Mycobacterium tuberculosis could be determined within 2 h by using pyrosequencing-based approach. Pyrosequencing results of rifampin-resistant (n = 21) and rifampin-susceptible (n = 20) M. tuberculosis isolates were concordant with those based on drug susceptibility testing and conventional DNA sequencing. Results showed pyrosequencing-based approach is a rapid, sensitive, and efficient detection method of rifampin-resistant M. tuberculosis.

Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology.

AIM: To develop a real-time PCR for detecting hepatitis B virus (HBV) DNA based on TaqMan technology using a new MGB probe. METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured. RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 10(0) and 10(9) DNA copies/reaction (r>0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy. CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.

[Quantification of mRNA expression for angiotensinogen in rat arterial tissues by real-time PCR].

AIM: To develop a real-time PCR method for quantifying low abundance mRNA expression in rat arterial tissues and examine differential changes of angiotensinogen (AGT) gene expression in basilar and femoral arterial tissues after 8 weeks simulated weightlessness. METHODS: After 8-wk of simulation, basilar and femoral arteries were harvested from both simulated weightless (SUS) and control (CON) rats. Then total RNA was extracted and reverse transcribed. The real-time PCR using TaqMan-MGB probe was performed to quantify AGT mRNA expression. The amplification efficiency (E) of PCR was calculated from the slope of standard curve. The threshold cycle (Ct) was detected by changes in fluorescence during a real-time PCR. Finally, the relative expression ratio of the target gene (AGT) to the reference gene (GAPDH) was calculated using E and Ct according to the mathematical model derived from the equation calculating the starting fluorescence (Ro). RESULTS: After a 8-weeks simulated weightlessness, the AGT mRNA expression increased by 240 percent in basilar arterial, and decreased by 66 percent in femoral arterial tissues. CONCLUSION: The specificity, sensitivity, precision, reproducibility, and simplicity of real-time PCR method using TaqMan-MGB probe make it particularly suitable for quantification of low abundance mRNA in arterial tissue from rats.

[Detection of KatG 315 gene mutation associated with isoniazid resistance in Mycobacterium tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection technology].

OBJECTIVE: To study the relationship between KatG 315 mutation and isoniazid (INH) resistance in M. tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP), and to develop a new method that can detect INH-resistant M. tuberculosis precisely and quickly. METHODS: Isolates of M. tuberculosis resistant to INH from 82 tuberculosis patients were cultured on culture medium with different INH concentrations. DNA of all M. tuberculosis samples was extracted and amplification of 271 bp KatG gene fragment was carried out by polymerase chain reaction (PCR). After digestion of the excess primers and dNTPs in PCR products by clean-up reagents, template-directed dye-terminator incorporation reaction was performed, and the Acyclo-terminators labeled Rhodamine 110 (R110) or 6-carboxytetramethylrhodamine (TAMRA) were incorporated into the detection primers specifically. Then fluorescence polarization value was measured using Victor2 multi-label counter and the genotypes of the mutation site in 315 condon of KatG gene of all samples were investigated. RESULTS: There were 15 out of the 29 isolates of M. tuberculosis with high INH-resistance showed G-->C mutation in 315 condon of KatG gene, including 4 isolates with both mutated and non-mutated trains, the mutation frequency being 52%. In 32 isolates of M. tuberculosis with low INH-resistance, 15 were mixed infection, the mutation frequency being 47%. No mutation in 315 condon of KatG gene was found in 21 susceptible isolates of M. tuberculosis. CONCLUSIONS: The G-->C mutation in 315 condon of KatG gene was associated with INH-resistance of M. tuberculosis. TDI-FP technology was a reliable, easy to use and high-through method to detect mutation of KatG gene. It may be a useful technique for the diagnosis of INH-resistance in the future.

Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization.

AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients. METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system. The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and R110 or TAMRA labeled acyclo-terminator was added on the 3' end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed. RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9%), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD phenotype (7.1%). CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.