Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility.

Due to its decade-long progression, colorectal cancer (CRC) is most suitable for population screening to achieve a significant reduction in its incidence and mortality. DNA methylation has emerged as a potential marker for the early detection of CRC. However, the current mainstream methylation detection method represented by bisulfite conversion has issues such as tedious operation, DNA damage, and unsatisfactory sensitivity. Herein, a new high-performance CRC screening tool based on the promising specific terminal-mediated polymerase chain reaction (STEM-PCR) strategy is developed. CRC-related methylation-specific candidate CpG sites are first prescreened through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases using self-developed bioinformatics. Next, 9 homebrew colorectal cancer DNA methylated STEM‒PCR assays (ColoC-mSTEM) with high sensitivity (0.1%) and high specificity are established to identify candidate sites. The clinical diagnostic performance of these selected methylation sites is confirmed and validated by a case-control study. The optimized diagnostic model has an overall sensitivity of 94.8% and a specificity of 95.0% for detecting early-stage CRC. Taken together, ColoC-mSTEM, based on a single methylation-specific site, is a promising diagnostic approach for the early detection of CRC which is perfectly suitable for the screening needs of CRC in primary healthcare institutions.

NMDA receptor within nucleus accumbens shell regulates propofol self-administration through D1R/ERK/CREB signalling pathway.

Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions between N-methyl-D-aspartate receptor (NMDAR) and the dopamine D1 receptor (D1R), as well as their downstream ERK/CREB signalling pathway in the NAc, are integral in regulating reward-seeking behaviour. Nevertheless, it remains unclear whether NMDARs and the NMDAR-D1R/ERK/CREB signalling pathway in the NAsh are involved in mediating propofol addiction. To investigate it, we conducted experiments with adult male Sprague-Dawley rats to establish a model of propofol self-administration behaviour. Subsequently, we microinjected D-AP5 (a competitive antagonist of NMDARs, 1.0-4.0 µg/0.3 µL/site) or vehicle into bilateral NAsh in rats that had previously self-administered propofol to examine the impact of NMDARs within the NAsh on propofol self-administration behaviour. Additionally, we examined the protein expressions of NR2A and NR2B subunits, and the D1R/ERK/CREB signalling pathways within the NAc. The results revealed that propofol administration behaviour was enhanced by D-AP5 pretreatment in NAsh, accompanied by elevated expressions of phosphorylation of NR2A (Tyr1246) and NR2B (Tyr1472) subunits. There were statistically significant increases in the expressions of D1Rs, as well as in the phosphorylated ERK1/2 (p-ERK1/2) and CREB (p-CREB). This evidence substantiates a pivotal role of NMDARs in the NAsh, with a particular emphasis on the NR2A and NR2B subunits, in mediating propofol self-administration behaviour. Furthermore, it suggests that this central reward processing mechanism may operate through the NMDAR-D1R/ERK/CREB signal transduction pathway.

Platelet membrane-derived biomimetic microbubbles with enhanced targeting ability for the early detection of myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a widely recognized cardiovascular disease that significantly impacts the prognosis of patients undergoing myocardial infarction recanalization. This condition can be fatal and involves complex pathophysiological mechanisms. Early diagnosis of MIRI is crucial to minimize myocardial damage and reducing mortality. Based on the inherent relationship between platelets and MIRI, we developed biomimetic microbubbles coated with platelet membrane (MB-pla) for early identification of MIRI. The MB-pla were prepared through a recombination process involving platelet membrane obtained from rat whole blood and phospholipids, blended in appropriate proportions. By coating the microbubbles with platelet membrane, MB-pla acquired various adhesion molecules, thereby gaining the capability to selectively adhere to damaged endothelial cells in the context of MIRI. In vitro experiments demonstrated that MB-pla exhibited remarkable targeting characteristics, particularly toward type IV collagen and human umbilical vein endothelial cells that had been injured through hypoxia/reoxygenation procedures. In a rat model of MIRI, the signal intensity produced by MB-pla was notably higher than that of control microbubbles. These findings were consistent with results obtained from fluorescence imaging of isolated hearts and immunofluorescence staining of tissue sections. In conclusion, MB-pla has great potential as a non-invasive early detection method for MIRI. Furthermore, this approach can potentially find application in other conditions involving endothelial injury in the future.

Ursolic acid reduces oxidative stress injury to ameliorate experimental autoimmune myocarditis by activating Nrf2/HO-1 signaling pathway.

Background: Oxidative stress is crucial in experimental autoimmune myocarditis (EAM)-induced inflammatory myocardial injury. Ursolic acid (UA) is an antioxidant-enriched traditional Chinese medicine formula. The present study aimed to investigate whether UA could alleviate inflammatory cardiac injury and determine the underlying mechanisms. Methods: Six-week-old male BALB/c mice were randomly assigned to one of the three groups: Sham, EAM group, or UA intervention group (UA group) by gavage for 2 weeks. An EAM model was developed by subcutaneous injection of α-myosin heavy chain derived polypeptide (α-MyHC peptide) into lymph nodes on days 0 and 7. Echocardiography was used to assess cardiac function on day 21. The inflammation level in the myocardial tissue of each group was compared using hematoxylin and eosin staining (HE) of heart sections and Interleukin-6 (IL-6) immunohistochemical staining. Masson staining revealed the degree of cardiac fibrosis. Furthermore, Dihydroethidium staining, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to determine the mechanism of cardioprotective effects of UA on EAM-induced cardiac injury, and the level of IL-6, Nrf2, and HO-1. Results: In EAM mice, UA intervention significantly reduced the degree of inflammatory infiltration and myocardial fibrosis while improving cardiac function. Mechanistically, UA reduced myocardial injury by inhibiting oxidative stress (as demonstrated by a decrease of superoxide and normalization of pro- and antioxidant enzyme levels). Interestingly, UA intervention upregulated the expression of antioxidant factors such as Nrf2 and HO-1. In vitro experiments, specific Nrf2 inhibitors reversed the antioxidant and antiapoptotic effects of ursolic acid, which further suggested that the amelioration of EAM by UA was in a Nrf2/HO-1 pathway-dependent manner. Conclusion: These findings indicate that UA is a cardioprotective traditional Chinese medicine formula that reduces EAM-induced cardiac injury by up-regulating Nrf2/HO-1 expression and suppressing oxidative stress, making it a promising therapeutic strategy for the treatment of EAM.

Sequence terminus dependent PCR for site-specific mutation and modification detection.

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).

Sensitive GlaI digestion and terminal transferase PCR for DNA methylation detection.

Highly sensitive and specific detection of DNA methylation is critical for early diagnosis and therapy of cancer. Herein, we propose a novel bisulfite-free PCR assay based on a GlaI methylation specific digestion and terminal transferase (TdT) extension for the detection of methylated DNA with high sensitivity and specificity, denoted as GlaI-TdT methylation PCR. For GlaI-TdT methylation PCR assay, the methylated CpG site is recognized and cut by GlaI selectively firstly, leading to the generation of product with specific free 3' end. The free 3' end can be further extended with TdT and served as template for the followed quantitative PCR. The specificity of GlaI-TdT methylation PCR depends on the specific methylation discrimination of GlaI and the existence of poly-T sequence as the extension of TdT. The sensitivity of GlaI-TdT PCR for methylated DNA can achieve 10 copies/reaction with 10,000 copies unmethylated background. The detection performance of GlaI-TdT methylation PCR was also evaluated using colorectal cancer tissue samples, with the results shown great accordance with standard bisulfite-PCR sequencing. Based on its high sensitivity, high specificity, simple and convenient, GlaI-TdT methylation PCR has the great potential to become a promising and robust bisulfite-free procedure for the detection of DNA methylations.

Preparation and characterization of layer-by-layer hypoglycemic nanoparticles with pH-sensitivity for oral insulin delivery.

New self-assembled layer-by-layer nanoparticles, based on hypoglycemic, mucoadhesive, epithelium-penetrating and pH-sensitive functional materials, have been synthesized. The confirmed hypoglycemic and negatively charged polygalacturonic acid, the well-known cell penetrating and positively charged chitosan, and the negatively charged, pH-sensitive polymer alginate were the components of these nanoparticles. The electrical attraction between the negatively charged PGLA and alginate and the positively charged chitosan motivated the assembly and supported the pH-sensitivity of the nanoparticles. The LBL4 and LBL4-Alg nanoparticles were characterized in vitro and in vivo. The pH-sensitivity of LBL4-Alg nanoparticles was greater, with the turning point near pH = 5. Moreover, according to the pharmacological and pharmacokinetic studies, the significant hypoglycemic effect was observed only 1 h after gavage with LBL4 and LBL4-Alg nanoparticles. Insulin was continually released for at least 6 h. These results show that the new self-assembled LBL4-Alg nanoparticles with pH-sensitivity have great potential for oral insulin delivery.

Preparation and Characterization of Hypoglycemic Nanoparticles for Oral Insulin Delivery.

Polymeric nanoparticles have been widely investigated as insulin delivery systems for oral administration. However, the toxic nature of many artificial polymers hampers their effective application, creating a demand for the further exploration of alternative natural polymers. In addition, ethnobotanical research has reported that over 800 plant species have a hypoglycemic function, some of which are polymers. For the advantages of both areas to be combined, the aim of this work was to choose an organic hypoglycemic polymer and prepare it into an insulin carrier to build a dual-functional oral insulin delivery system. We found that the insulin loading rate, release mode, thermostability, and both in vitro and in vivo absorption and efficacy varied with the different modifications of polygalacturonic acid (PGLA) nanoparticulate backbones. By in vivo pharmaceutical testing and constantly monitoring the symptoms of type 1 diabetic (T1D) rats, we ascertained the hypoglycemic function of the nanoparticles and showed that overall diabetic symptoms were ameliorated after the long-term daily administration of nanoparticles with no significant damage to organ structure or cell viability.

Molecular characterization and expression analysis of the critical floral genes in hickory (Carya cathayensis Sarg.).

The full ORFs of three floral genes in hickory (Carya cathayensis Sarg.), CcAGL24 (the AGAMOUS-LIKE24 homolog), CcSOC1 (the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 homolog) and CcAP1 (the APETALA1 homolog) are derived using a 5' RACE PCR protocol. Through sequence alignment and phylogenetic analysis, it is demonstrated that the three genes belong to the MADS-Box family. According to the evolutionary trees of the three genes, the homologous genes from the same family cluster well together, while those from different orders doesn't match evolutionary regularity of individual organisms. The result of Quantitative RT-PCR analysis shows that the transcriptional levels of the three genes are up-regulated in early stage and down-regulated in late stage in pistillate floral development. However, it takes different time to reach respective expression peak among the three genes. In staminate floral development, the transcription trend of the three genes is up-regulated, subsequently down-regulated, and then up-regulated again. Nevertheless, those trajectories, peaks, expression levels, inflection points are different in pistillate floral development. The result suggests that their functions are different in between pistillate and staminate floral development. The probable ordinal site of the three genes in the flowering network from top down is CcAGL24, CcSOC1, and CcAP1, which is identical to that in herbaceous plants. Moreover, several adverse environmental factors trigger several negative genes and then confine the development of staminate floral buds. Our results suggest the possible relationship among the three critical floral genes and their functions throughout the floral development in hickory.