A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation.

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3' end and 5' end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1-H2 duplex. Then 3' terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5' terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3' end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes.

In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.

Universal point-of-care detection of proteins based on proximity hybridization-mediated isothermal exponential amplification.

We have developed a point-of-care (POC) lateral flow biosensor (LFB) for universal protein detection based on a proximity hybridization-mediated protein-to-DNA signal transducer, isothermal exponential amplification (EXPAR) and catalytic hairpin assembly (CHA) with high sensitivity and specificity. In this assay, a protein signal transducer was employed to convert the input protein to the output DNA signal. Antibody conjugated DNA1 was firstly hybridized with the output DNA (DNA3). The binding of antibody conjugated DNA1 and DNA2 to the same protein was able to increase the local concentrations, resulting in strand displacement between DNA3 and DNA2. DNA3 with nicking endonuclease recognition sequences at the 5' end then hybridized with hairpin probe 1 to mediate EXPAR in the presence of nicking endonuclease and polymerase. A large number of single strand DNA were produced in the circle of nicking, polymerization and strand displacement. The resulting ssDNA products were further amplified by CHA to generate double-stranded DNA products. The double-stranded DNA products were detected with a lateral flow biosensor within 5 min. This proposed assay has very high sensitivity and selectivity with a dynamic response ranging from 1 fM to 100 nM, and the detection limit was 0.74 fM. This work provides a universal and simple method for protein detection.

Association between ADAMTS7 TagSNPs and the risk of myocardialinfarction.

PURPOSE OF THE STUDY: Genome-wide association studies have revealed an association of ADAMTS7 polymorphisms with the risk of cardiovascular diseases. Nonetheless, the role of ADAMTS7 polymorphisms on myocardial infarction (MI) risk remains poorly understood. Here, we aim to evaluate the effect of ADAMTS7 tag single nucleotide polymorphisms (SNPs) on individual susceptibility to MI. STUDY DESIGN: Genotyping of the four tagSNPs (rs1994016, rs3825807, rs4380028 and rs7173743) was performed in 232 MI cases and 661 control subjects using PCR-ligase detection reaction (LDR) method. The association of these four tagSNPs with MI risk was performed with SPSS software. RESULTS: Multivariate logistic regression analysis showed that ADAMTS7 tagSNP rs3825807 exhibited a significant effect on MI risk. Compared with the TT homozygotes, the CT genotype (OR1.93, 95% CI1.30to 2.85, Pc=0.004) and the combined CC/CT genotypes (OR1.70, 95% CI1.16 to 2.50, Pc=0.028) were statistically significantly associated with the increased risk for MI. Further stratified analysis revealed a more significant association with MI risk among older subjects, hypertensives, non-diabetics and patients with hyperlipidaemia. Consistently, the haplotype rs1994016T-rs3825807C containing rs3825807 C allele exhibited increased MI risk (OR1.52, 95% CI1.10 to 2.10, p=0.010). However, we did not detect any association of the other three tagSNPs with MI risk. CONCLUSIONS: Our finding suggest that ADAMTS7 tagSNP rs3825807 contributes to MI susceptibility in the Chinese Han population. Further studies are necessary to confirm the general validity of our findings and to clarify the underlying mechanism for this association.

Genetic susceptibility of five tagSNPs in the endothelin-1 (EDN1) gene to coronary artery disease in a Chinese Han population.

Endothelin-1 (ET-1) plays important roles in endothelial dysfunction, vascular physiology, inflammation, and atherosclerosis. Nonetheless, the role of ET-1 (EDN1) gene variants on coronary artery disease (CAD) risk remains poorly understood. The aim of the present study was to evaluate the role of EDN1 gene polymorphisms on individual susceptibility to CAD. We genotyped five tagSNPs (single-nucleotide polymorphisms) (rs6458155, rs4145451, rs9369217, rs3087459, and rs2070699) within EDN1 gene in 525 CAD patients and 675 control subjects. In a multivariate logistic regression analysis, we detected an association of rs6458155 in EDN1 gene with the CAD risk; compared with the TT homozygotes, the CT heterozygotes (odds ratio (OR) = 1.53, 95% confidence interval (CI) = 1.02-2.29, P=0.040) and the CC homozygotes (OR = 1.55, 95% CI = 1.01-2.36, P=0.043) were statistically significantly associated with the increased risk for CAD. A similar trend of the association was found in dominant model (OR = 1.53, 95% CI = 1.05-2.25, P=0.029). Consistently, the haplotype rs6458155C-rs4145451C containing rs6458155 C allele exhibited the increased CAD risk (OR = 1.22, 95% CI = 1.03-1.43, and P=0.018). In addition, CT genotype of rs6458155 conferred the increased plasma ET-1 levels compared with TT genotype (P<0.05). No association of the other four tagSNPs in EDN1 gene with CAD risk was observed. In conclusion, our study provides the first evidence that EDN1 tagSNP rs6458155 is associated with CAD risk in the Chinese Han population, which is probably due to the influence of the circulating ET-1 levels.

Matrix-induced autologous chondrocyte implantation for the treatment of chondral defects of the knees in Chinese patients.

Articular cartilage injury is the most common type of damage seen in clinical orthopedic practice. The matrix-induced autologous chondrocyte implant (MACI) was developed to repair articular cartilage with an advance on the autologous chondrocyte implant procedure. This study aimed to evaluate whether MACI is a safe and efficacious cartilage repair treatment for patients with knee cartilage lesions. The primary outcomes were the Knee Injury and Osteoarthritis Outcome Score (KOOS) domains and magnetic resonance imaging (MRI) results, compared between baseline and postoperative months 3, 6, 12, and 24. A total of 15 patients (20 knees), with an average age of 33.9 years, had a mean defect size of 4.01 cm(2). By 6-month follow-up, KOOS results demonstrated significant improvements in symptoms and knee-related quality of life. MRI showed significant improvements in four individual graft scoring parameters at 24 months postoperatively. At 24 months, 90% of MACI grafts had filled completely and 10% had good-to-excellent filling of the chondral defect. Most (95%) of the MACI grafts were isointense and 5% were slightly hyperintense. Histologic evaluation at 15 and 24 months showed predominantly hyaline cartilage in newly generated tissue. There were no postoperative complications in any patients and no adverse events related to the MACI operation. This 2-year study has confirmed that MACI is safe and effective with the advantages of a simple technique and significant clinical improvements. Further functional and mechanistic studies with longer follow-up are needed to validate the efficacy and safety of MACI in patients with articular cartilage injuries.

Posterolateral spinal fusion with nano-hydroxyapatite-collagen/PLA composite and autologous adipose-derived mesenchymal stem cells in a rabbit model.

Spinal fusion is routinely performed to treat low back pain caused by degeneration of intervertebral discs. An autologous bone graft derived from the iliac crest is the standard procedure used for spinal fusion. However, several shortcomings, including pseudarthrosis, pain and the need for blood transfusion are known to be associated with the procedure. Our study analysed the effectiveness of a new mineralized collagen matrix, nano-hydroxyapatite-collagen-polylactic acid (nHAC-PLA), combined with autologous adipose-derived mesenchymal stem cells (ADMSCs) as a graft material for posterolateral spinal fusion in a rabbit model. Forty rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC-PLA composite group (nHAC-PLA), autologous iliac crest bone mixed with nHAC-PLA composite group (ACB + nHAC-PLA), and nHAC-PLA composite combined with ADMSCs (ADMSCs + nHAC-PLA). The viability and the proliferation of the ADMSCs seeded on the scaffolds were evaluated by live/dead kit and MTT assay in vitro, respectively. Lumbar posterolateral fusions were assessed by manual palpation, radiographical and histological procedures, mechanical strength and scanning electronic microscopy (SEM) in 10 weeks of observation. The results showed that the rate of fusion was significantly higher in the ACB and ADMSCs + nHAC-PLA groups than that in the nHAC-PLA and ACB + nHAC-PLA groups. It was not significantly higher in the ACB group than in the ADMSCs + nHAC-PLA group. From microstructural analysis of the samples using histological staining methods, there was more new bone-like tissue formation in the ACB and ADMSCs + nHAC-PLA groups than that in the other two groups at the 10th postoperative week. Our study demonstrated the effective impact of nHAC-PLA combined with ADMSCs in rabbit posterolateral spinal fusion.