Rapid in situ RNA imaging based on Cas12a thrusting strand displacement reaction.

RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

Isomerization and Stabilization of Amygdalin from Peach Kernels.

In this study, isomerization conditions, cytotoxic activity, and stabilization of amygdalin from peach kernels were analyzed. Temperatures greater than 40 °C and pHs above 9.0 resulted in a quickly increasing isomer ratio (L-amygdalin/D-amygdalin). At acidic pHs, isomerization was significantly inhibited, even at high temperature. Ethanol inhibited isomerization; the isomer rate decreased with the ethanol concentration increasing. The growth-inhibitory effect on HepG2 cells of D-amygdalin was diminished as the isomer ratio increased, indicating that isomerization reduces the pharmacological activity of D-amygdalin. Extracting amygdalin from peach kernels by ultrasonic power at 432 W and 40 °C in 80% ethanol resulted in a 1.76% yield of amygdalin with a 0.04 isomer ratio. Hydrogel beads prepared by 2% sodium alginate successfully encapsulated the amygdalin, and its encapsulation efficiency and drug loading rate reached 85.93% and 19.21%, respectively. The thermal stability of amygdalin encapsulated in hydrogel beads was significantly improved and reached a slow-release effect in in vitro digestion. This study provides guidance for the processing and storage of amygdalin.

Identification of four genes and biological characteristics of esophageal squamous cell carcinoma by integrated bioinformatics analysis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has become one of the most serious diseases affecting populations worldwide and is the primary subtype of esophageal cancer (EC). However, the molecular mechanisms governing the development of ESCC have not been fully elucidated. METHODS: The robust rank aggregation method was performed to identify the differentially expressed genes (DEGs) in six datasets (GSE17351, GSE20347, GSE23400, GSE26886, GSE38129 and GSE77861) from the Gene Expression Omnibus (GEO). The Search Tool for the Retrieval of Interacting Genes (STRING) database was utilized to extract four hub genes from the protein-protein interaction (PPI) network. Module analysis and disease free survival analysis of the four hub genes were performed by Cytoscape and GEPIA. The expression of hub genes was analyzed by GEPIA and the Oncomine database and verified by real-time quantitative PCR (qRT-PCR). RESULTS: In total, 720 DEGs were identified in the present study; these genes consisted of 302 upregulated genes and 418 downregulated genes that were significantly enriched in the cellular component of the extracellular matrix part followed by the biological process of the cell cycle phase and nuclear division. The primary enriched pathways were hsa04110:Cell cycle and hsa03030:DNA replication. Four hub genes were screened out, namely, SPP1, MMP12, COL10A1 and COL5A2. These hub genes all exhibited notably increased expression in ESCC samples compared with normal samples, and ESCC patients with upregulation of all four hub genes exhibited worse disease free survival. CONCLUSIONS: SPP1, MMP12, COL10A1 and COL5A2 may participate in the tumorigenesis of ESCC and demonstrate the potential to serve as molecular biomarkers in the early diagnosis of ESCC. This study may help to elucidate the molecular mechanisms governing ESCC and facilitate the selection of targets for early treatment and diagnosis.

Risk factors for complications of therapeutic endoscopy for upper gastrointestinal subepithelial lesions. / ä¸Šæ¶ˆåŒ–é“ä¸Šçš®ä¸‹ç— å˜è¡Œå† é•œæ²»ç–—åŽå¹¶å‘ç—‡çš„å±é™©å› ç´ .

OBJECTIVES: To study the risk factors for complications after endoscopic therapy for upper gastrointestinal subepithelial lesions (SELs). METHODS: Retrospective analysis was performed on 184 patients in the Department of Gastroenterology in the Third Xiangya Hospital, Central South University after therapeutic endoscopy [endoscopic submucosal dissection (ESD), endoscopic full-thickness resection (EFR), endoscopic submucosal excavation (ESE), and submucosal tunneling endoscopic resection (STER)] for the upper gastrointestinal SELs from 2014-09-01 to 2019-09-30. The clinic data were collected and risk factors for postoperative complications were analyzed. RESULTS: Among the 184 patients, 22 patients were in the complication group (including 3 cases of delayed bleeding, 2 cases of delayed perforation, and 17 cases of electrocoagulation syndrome) and 162 patients were in the non-complication group. There was no significant difference between the complication group and the non-complication group in gender, age over 70 year, basic diseases, lesion location, lesion invasion layers, pathological results, endoscopic therapy, and preventive closure of wounds (all P>0.05). The differences between the two groups in lesion diameter over 40 mm, operative time over 120 minutes, and rate of intraoperative perforation were significant (all P<0.05). Logistic regression analysis showed that lesion diameter over 40 mm and operative time over 120 minutes were independent risk factors for postoperative complications. CONCLUSIONS: For the patients with upper gastrointestinal SELs after endoscopic minimally invasive therapy with the lesion diameter over 40 mm and the operative time over 120 minutes, it needs to highly alert to the occurrence of postoperative complications.

Risk of rebleeding from gastroesophageal varices after initial treatment with cyanoacrylate; a systematic review and pooled analysis.

BACKGROUND: Cyanoacrylate alone or in combination with other interventions, can be used to achieve variable rates of success in preventing rebleeding. Our study aims to assess the pooled risk of gastric and esophageal varices rebleeding after an initial treatment with cyanoacrylate alone and/or in combination with other treatments, by a systematic review of the literature and pooled analysis. METHODS: PubMed, EMBASE, SCOPUS, and the Cochrane library were searched for studies that reported the risk of rebleeding during the follow-up period after treatment of gastric or esophageal varices with either cyanoacrylate alone or in combination with other treatments. Standard error, upper and lower confidence intervals at 95% confidence interval for the risk were obtained using STATA Version 15 which was also used to generate forest plots for pooled analysis. The random or fixed effect model was applied depending on the heterogeneity (I2). RESULTS: A total of 39 studies were found to report treatment of either gastric or esophageal varices with either cyanoacrylate alone or in combination with other treatments. When gastric varices are treated with cyanoacrylate alone, the risk of rebleeding during the follow-up period is 0.15(Confidence Interval: 0.11-0.18). When combined with lipiodol; polidocanol or sclerotherapy the rebleeding risks are 0.13 (CI:0.03-0.22), 0.10(CI:0.02-0.19), and 0.10(CI:0.05-0.18), respectively. When combined with percutaneous transhepatic variceal embolization; percutaneous transhepatic variceal embolization; endoscopic ultrasound guided coils; or with ethanolamine, the rebleeding risk are 0.10(CI:0.03-0.17), 0.10(CI:0.03-0.17), 0.07(CI:0.03-0.11) and 0.08(CI:0.02-0.14), respectively. When esophageal varices are treated with cyanoacrylate alone, the risk of rebleeding is 0.29(CI:0.11-0.47). When combined with percutaneous transhepatic variceal embolization; sclerotherapy; or band ligation, the risks of rebleeding are 0.16(CI:0.10-0.22), 0.12(CI:0.04-0.20) and 0.10(CI:0.04-0.24), respectively. When combined with a transjugular intrahepatic portosystemic shunt; or ethanolamine, the risks of rebleeding are 0.06(CI: - 0.01-0.12) and 0.02 (CI: - 0.02-0.05), respectively. CONCLUSION: In treating both gastric and esophageal varices, cyanoacrylate produces better results in terms of lower risk of rebleeding when combined with other treatments than when used alone. The combination of cyanoacrylate with ethanolamine or with endoscopic ultrasound guided coils produces the lowest risk of rebleeding in esophageal and gastric varices, respectively. We call upon randomized trials to test these hypotheses.

Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification.

An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2- causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.

[Clinical application of mammography-guided wire localization plus ultrasound-guided core-needle biopsy for breast microcalcification].

OBJECTIVE: To evaluate the clinical usefulness of mammography-guided wire localization plus ultrasound-guided core-needle biopsy of breast microcalcification and avoid the effects of preoperative excisional biopsy for patients undergoing sentinel node biopsy. METHODS: A total of 38 patients with unpalpable lesions, ultrasonic negativity and abnormal mammography received a guide wire under mammography and performed ultrasound-guided core-needle biopsy of breast microcalcification before preoperative excisional biopsy. RESULTS: All 38 lesions were successfully located and biopsied for pathological examination. Carcinoma was present in 9 lesions: ductal carcinoma in situ (DCIS) (6/38) and intraductal carcinoma with early infiltration (3/38). For 29 benign lesions, there were mastopathies (25/38), atypical ductal hyperplasia (ADH)(3/38) and intraductal papiloma (1/38). Breast microcalcification was detected in all lesions by ultrasound-guided core-needle biopsy. The detection rate of breast cancer detected was 30.8% and the diagnostic accuracy 100%. CONCLUSIONS: For patients with unpalpable lesions, ultrasound negativity and abnormal mammography, mammography-guided wire-localization plus ultrasound-guided core-needle biopsy of breast microcalcification is accurate, safe and feasible. And it may avoid the effects of preoperative excisional biopsy for patients with sentinel node biopsy. The procedure is worthy of wider clinical applications.

Reduced succinate dehydrogenase B expression is associated with growth and de-differentiation of colorectal cancer cells.

Succinate dehydrogenases (SDH), including SDHA, SDHB, SDHC, and SDHD, form the respiratory complex II in the mitochondria and play an important role in cell growth and homeostasis. In order to evaluate the expression and functional significance of SDH in colorectal cancer, the expression of four SDH subunits was analyzed, and SDHB protein was found to be significantly lower in colorectal cancer tissues. In vitro experiments including cell growth assay, colony formation assay, cell cycle analysis, and nude mouse xenograft of SDHB-transfected colorectal cancer cell line SW620 were performed. Notably, reduced SDHB expression in tumor tissues was associated with tumor de-differentiation, and restoration of SDHB could inhibit the growth of cancer cells both in vitro and in vivo. Furthermore, cDNA microarray of SDHB-transfected cell line showed that most of the differentially expressed genes are related to cell cycle control and cell proliferation. Thus, we conclude that SDHB expression is significantly decreased in human colorectal cancer tissues, and reconstitution of SDHB in colorectal cancer may be a potential therapeutic approach to inhibit aggressiveness of colorectal cancer.

Dynamic changes of peritoneal macrophages and subpopulations during ulcerative colitis to metastasis of colorectal carcinoma in a mouse model.

OBJECTIVE AND DESIGN: Patients with ulcerative colitis have increased risk of colorectal carcinoma, but little is known about how peritoneal macrophages are involved in ulcerative colitis-associated carcinogenesis. We investigated the alteration of peritoneal macrophages and M1/M2 subpopulations during ulcerative colitis-associated carcinogenesis. MATERIALS AND METHODS: Expression and functional changes in peritoneal macrophages and M1/M2 subpopulations were investigated by histopathology, flow cytometry, immunofluorescence, cytokines expression by ELISA and QRT-PCR in an azoxymethane (AOM)- and dextran sodium sulfate (DSS)-induced chemical colitis-associated carcinoma mouse model using male Crj:CD-1 (ICR) mice. RESULTS: Striking evidence observed in histopathology, flow cytometry, cytokine detection, and gene expression analysis all revealed that inflammation-associated cytokines (IL-1ß, IL-10, IL-12, IL-6, TNF-α) and migration/invasion-associated factors (G-CSF, GM-CSF, CXCR4, VEGF, TGF-ß, ICAM-1) induced by peritoneal M2 macrophages increased significantly during the progression from inflammatory hyperplasia to carcinoma and metastasis. Similar functional changes occurred during peritoneal metastasis in M1 macrophages without changed polarization. CONCLUSIONS: These results suggested that peritoneal M2 macrophages played a critical role in ulcerative colitis-associated carcinogenesis, including unbalanced pro-inflammatory and anti-inflammatory axis and enhanced expression of migration/invasion-associated factors. Furthermore, functional changes of M1 macrophages occurred without changed polarization during carcinogenesis and metastasis.

Global, regional and national burden of inflammatory bowel disease in 204 countries and territories from 1990 to 2019: a systematic analysis based on the Global Burden of Disease Study 2019.

OBJECTIVES: We aimed to provide the most updated estimates on the global burden of inflammatory bowel disease (IBD) to improve management strategies. DESIGN: We extracted data from the Global Burden of Disease (GBD) 2019 database to evaluate IBD burden with different measures in 204 countries and territories from 1990 to 2019. SETTING: Studies from the GBD 2019 database generated by population-representative data sources identified through a literature review and research collaborations were included. PARTICIPANTS: Patients with an IBD diagnosis. OUTCOMES: Total numbers, age-standardised rates of prevalence, mortality and disability-adjusted life-years (DALYs), and their estimated annual percentage changes (EAPCs) were the main outcomes. RESULTS: In 2019, there were approximately 4.9 million cases of IBD worldwide, with China and the USA having the highest number of cases (911 405 and 762 890 (66.9 and 245.3 cases per 100 000 people, respectively)). Between 1990 and 2019, the global age-standardised rates of prevalence, deaths and DALYs decreased (EAPCs=-0.66,-0.69 and -1.04, respectively). However, the age-standardised prevalence rate increased in 13 out of 21 GBD regions. A total of 147 out of 204 countries or territories experienced an increase in the age-standardised prevalence rate. From 1990 to 2019, IBD prevalent cases, deaths and DALYs were higher among females than among males. A higher Socio-demographic Index was associated with higher age-standardised prevalence rates. CONCLUSIONS: IBD will continue to be a major public health burden due to increasing numbers of prevalent cases, deaths and DALYs. The epidemiological trends and disease burden of IBD have changed dramatically at the regional and national levels, so understanding these changes would be beneficial for policy makers to tackle IBD.

Global, regional, and national burden of 10 digestive diseases in 204 countries and territories from 1990 to 2019.

Background: Digestive diseases are very common worldwide and account for considerable health care use and expenditures. However, there are no global population-based estimates of the disease burden and temporal trend of digestive diseases. Methods: Annual case numbers, age-standardized rates of prevalence, incidence, death, and disability-adjusted life-years (DALYs), and their estimated annual percentage changes (EAPCs) for digestive diseases between 1990 and 2019 were derived from the Global Burden of Disease, Injuries, and Risk Factors Study (GBD) 2019. The association between digestive disease burden and the sociodemographic index (SDI) was investigated. We also calculated DALYs attributable to risk factors that had evidence of causation with digestive diseases. Results: Globally, in 2019, there were 88.99 million DALYs due to digestive diseases (3.51% of global DALYs). Digestive diseases were the 13th leading cause of DALYs globally in 2019. Global digestive disease DALYs were highest in the middle SDI quintile and in South Asia and were higher in males than females in 2019. Cirrhosis and other chronic liver diseases constituted the highest proportion of categorized digestive disease DALY burdens globally. From 1990 to 2019, the global age-standardized DALY rate of digestive diseases decreased from 1570.35 in 1990 to 1096.99 in 2019 per 1,00,000 population, with the EAPC being -1.32 (95% confidence interval [CI] -1.36 to -1.27). In 2019, the largest contributor to digestive disease DALYs at the global level, for both sexes, was alcohol use. Conclusion: The results of this systematic analysis suggest that the global burden of digestive diseases is substantial and varies markedly according to age, sex, SDI, and geographical region. These results provide comprehensive and comparable estimates that can potentially inform efforts toward digestive disease control worldwide.

A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.

An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10-5 U µL-1 in the concentration scope from 1 × 10-4 U µL-1 to 1 × 10-1 U µL-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.

Global, regional and national burden of gastroesophageal reflux disease, 1990-2019: update from the GBD 2019 study.

BACKGROUND: Because trends in the epidemiology and burden of gastroesophageal reflux disease (GERD) are changing, reinvestigating the geographical differences and trend changes is essential. Here we evaluated the latest epidemiologic patterns and trends for GERD, using data from Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. METHODS: Annual case numbers, age-standardized rates of prevalence, incidence, and years of life lived with disability (YLDs), and their estimated annual percentage changes (EAPCs) for GERD between 1990 and 2019 were derived from the GBD 2019 study. Association between GERD burden and socio-demographic index (SDI) was also investigated. RESULTS: In 2019, there were 783.95 million cases of GERD globally. Between 1990 and 2019, the total number of prevalent cases, incident cases, and YLDs increased by 77.53%, 74.79%, and 77.19%, respectively. The global age-standardized incidence rate (ASIR) and age-standardized YLD rate (ASYR) increased during this period (EAPC = 0.06 and 0.05, respectively). Tropical Latin America and East Asia had the highest and lowest age-standardiZed prevalence rate (ASPR), ASIR, and ASYR in 2019, respectively. From 1990 to 2019, prevalent cases, incident cases, YLDs, and their corresponding age-standardized rates of GERD were higher in females than males in all years. Higher SDI was associated with lower ASPR, ASIR, and ASYR of GERD in 2019. CONCLUSIONS: GERD will continue to be a major public health burden due to increasing numbers of prevalent cases, incident cases, and YLDs. In order to tackle this troublesome disease, it is crucial to understand the changes in both global and regional trends in epidemiology and the burden for policymakers and other stakeholders. Key messagesThis is the most updated estimate on GERD epidemiology globally, including 204 countries, some of which were not assessed before.The overall burden of GERD continued to worsen with the prevalent cases increasing by 77.53% from 441.57 million in 1990 to 783.95 million in 2019.GERD is likely to remain a common reason for consultation in primary care, and our data may allow for health service provision planning.

Activating IL-6/STAT3 Enhances Protein Stability of Proteasome 20S α+ß in Colorectal Cancer by miR-1254.

A widely recognized feature of colorectal cancer (CRC) is an increase in cytokine levels, which result in an inflammatory environment in the tumor. Interleukin-6 (IL-6) is a robust protumor cytokine. Several studies suggest that IL-6 plays a role in the development of tumors. Most intracellular protein breakdown occurs in eukaryotes via the ubiquitin-proteasome pathway; this mechanism may also be involved in cancer pathogenesis. The tumor tissues and paracancerous tissues were collected from 90 patients with colorectal cancer. The expressions of pSTAT3, proteasome 20S α+ß, miR-1254, and PSMD1 in tissues were detected by immunohistochemistry, ELISA, and qRT-PCR, and the effects of pSTAT3 and proteasome 20s α+ß expressions on the survival of patients were studied. HCT116 and HCT116-R cells were cultured and added IL-6, AG490, STAT3 plasmid, or overexpression/knockdown of miR-1254 in cells. Immunofluorescence, western blot, qRT-PCR, double luciferase gene reporter assay, and flow cytometry were used to detect the expression of pSTAT3, STAT3, proteasome 20s α+ß, miR-1254, and PSMD1 and cell cycle. The nude mouse xenograft model was constructed and divided into 3 groups: PBS group, IL-6 treatment group, and IL-6+miR-1254 mimic group. After 28 days, the tumor tissues were collected, and the expressions of miR-1254, pSTST3, proteasome 20s α+ß, and PSMD1 in the tissues were detected by qRT-PCR and immunohistochemistry, respectively. Our study discovered that the level of proteasome 20S α+ß had a strong connection with pSTAT3 in CRC patients. They were also linked to the development and clinical outcome of CRC. In addition, we found that IL-6 dramatically increased the expression of proteasome 20S α+ß and pSTAT3; however, it did not affect the proteasome 20S α+ß mRNA synthesis. Circulating proteasome concentration correlated with tumor tissue proteasome 20S α+ß. STAT3 could occupy the miR-1254 promoter to inhibit transcription, and it could suppressed miR-1254 which targeted PSMD10, promoting proteasome 20S α+ß protein stability. This is a prospective target for developing a new colorectal cancer therapy strategy.

The global, regional and national burden of stomach cancer and its attributable risk factors from 1990 to 2019.

We aimed to estimate the incidence, mortality, and disability-adjusted life-years (DALYs) of stomach cancer at the global, regional, and national levels. Stomach cancer resulted in 1.3 million (1.2-1.4 million) incident cases, 9.5 hundred thousand (8.7-10.4 hundred thousand) deaths, and 22.2 million (20.3-24.1 million) DALYs in 2019. The age-standardized incidence rate, death rate and DALY rate were 15.6 (14.1-17.2), 11.9 (10.8-12.8), and 268.4 (245.5-290.6) per 100,000 person-years, respectively. Between 1990 and 2019, the global age-standardized incidence rate, death rate, and DALY rate decreased by - 30.5% (- 36.7 to - 22.9), - 41.9% (- 47.2 to - 36.3), and - 45.6% (- 50.8 to - 39.8), respectively. In 2019, most of the global numbers of incidence, death and DALYs were higher among males than females. A considerable burden of stomach cancer was attributable to smoking and a high-sodium diet. Although the global age-standardized incidence and death rates have decreased, continued growth in absolute numbers in some regions, especially in East Asia, poses a major global public health challenge. To address this, public health responses should be tailored to fit each country's unique situation. Primary and secondary prevention strategies with increased effectiveness are required to reduce the incidence and mortality of stomach cancer, particularly in populations with a high disease burden.

A dual identification strategy based on padlock ligation and CRISPR/Cas14a for highly specific detection of BRAF V600E mutation in clinical samples.

BRAF V600E mutation is a single-nucleotide variation (SNV) that is widely found in various cancers and has been demonstrated to have a strong association with the prognosis and development of some diseases. Thus, we developed a strategy based on rolling circle amplification (RCA) and CRISPR/Cas14a to meet the great need for detecting highly specific BRAF V600E mutation in fine-needle biopsy samples. In this study, a padlock probe was designed to recognize and trigger subsequent ligase chain reactions (LCR). And due to the Taq DNA ligase, a great number of ligated annular padlock probes were generated in the presence of BRAF V600E mutation, subsequently generating long repeated single-strand DNA by RCA. The obtained amplicons were activators triggering the trans-cleavage of CRISPR/Cas14a. CRISPR/Cas14a shows outstanding performance in identifying ssDNA with single base mutation, which significantly increases the specificity of mutation discrimination. Under the optimal conditions, our strategy can identify BRAF V600E mutation down to 0.307 fM with a wide linear range from 1 fM to 10 pM. On the other hand, the dual identification strategy endows the method with terrific specificity for the detection of SNV. Furthermore, our method has been successfully employed to identify BRAF V600E mutation in clinical fine-needle aspiration samples, proving great potential for ultra-specific identification of low abundance BRAF V600E mutation and providing a novel method for diagnosis and treatment of cancer.

Integrative Proteo-Genomic Analysis for Recurrent Survival Prognosis in Colon Adenocarcinoma.

Background: The survival prognosis is the hallmark of cancer progression. Here, we aimed to develop a recurrence-related gene signature to predict the prognosis of colon adenocarcinoma (COAD). Methods: The proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and genomic data from the cancer genomic maps [The Cancer Genome Atlas (TCGA)] dataset were analyzed to identify co-differentially expressed genes (cDEGs) between recurrence samples and non-recurrence samples in COAD using limma package. Functional enrichment analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was conducted. Univariate and multivariate Cox regressions were applied to identify the independent prognostic feature cDEGs and establish the signature whose performance was evaluated by Kaplan-Meier curve, receiver operating characteristic (ROC), Harrell's concordance index (C-index), and calibration curve. The area under the receiver operating characteristic (ROC) curve (AUROC) and a nomogram were calculated to assess the predictive accuracy. GSE17538 and GSE39582 were used for external validation. Quantitative real-time PCR and Western blot analysis were carried out to validate our findings. Results: We identified 86 cDEGs in recurrence samples compared with non-recurrence samples. These genes were primarily enriched in the regulation of carbon metabolic process, fructose and mannose metabolism, and extracellular exosome. Then, an eight-gene-based signature (CA12, HBB, NCF1, KBTBD11, MMAA, DMBT1, AHNAK2, and FBLN2) was developed to separate patients into high- and low-risk groups. Patients in the low-risk group had significantly better prognosis than those in the high-risk group. Four prognostic clinical features, including pathological M, N, T, and RS model status, were screened for building the nomogram survival model. The PCR and Western blot analysis results suggested that CA12 and AHNAK2 were significantly upregulated, while MMAA and DMBT1 were downregulated in the tumor sample compared with adjacent tissues, and in non-recurrent samples compared with non-recurrent samples in COAD. Conclusion: These identified recurrence-related gene signatures might provide an effective prognostic predictor and promising therapeutic targets for COAD patients.

An ultrasensitive biosensing platform for FEN1 activity detection based on target-induced primer extension to trigger the collateral cleavage of CRISPR/Cas12a.

Flap endonuclease 1 (FEN1), a structure-selective endonuclease essential for DNA replication and repair, has been considered as a new promising marker for early cancer diagnosis. However, reliable, sensitive and convenient biosensors for FEN1 detection are still technically challenging. Herein, a fluorometric biosensor based on target-induced primer extension to initiate the collateral cleavage of CRISPR/Cas12a has been established for ultrasensitive and specific detection of FEN1 activity. Using branched DNA to probe FEN1 activity, the cleaved 5' flap initiated DNA polymerase-mediated primer extension to produce plenty of DNA duplexes containing protospacer adjacent motif (PAM) which act as activators to initiate the collateral cleavage activity of Cas12a protein, producing an significantly amplified fluorescence response for ultrasensitive determination of FEN1 activity. The developed biosensing platform displays excellent analytical performance, with a limit of detection (LOD) down to 8.9 × 10-5 U µL-1, and a wide linear range from 1.0 × 10-4 to 5.0 × 10-1 U µL-1. Moreover, the proposed strategy was successfully used for FEN1 detection in serums and cell lysates and suggests potential clinical applications, which may provide a reliable approach for FEN1 that will allow effective diagnosis in the early stages of related cancer.

Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 µM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 µM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.