Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration.

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403-1409 in the HMGB1 3' UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and ß-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

dbCNV: deleteriousness-based model to predict pathogenicity of copy number variations.

BACKGROUND: Copy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features. RESULTS: From single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs. CONCLUSION: The performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.

Musashi-2 Deficiency Triggers Colorectal Cancer Ferroptosis by Downregulating the MAPK Signaling Cascade to Inhibit HSPB1 Phosphorylation.

BACKGROUND: Musashi-2 (MSI2) is a critical RNA-binding protein (RBP) whose ectopic expression drives the pathogenesis of various cancers. Accumulating evidence suggests that inducing ferroptosis of tumor cells can inhibit their malignant biological behavior as a promising therapeutic approach. However, it is unclear whether MSI2 regulates cell death in colorectal cancer (CRC), especially the underlying mechanisms and biological effects in CRC ferroptosis remain elusive. METHODS: Experimental methods including qRT‒PCR, immunofluorescence, flow cytometry, western blot, co-immunoprecipitation, CCK-8, colony formation assay, in vitro cell transwell migration and invasion assays, in vivo xenograft tumor experiments, liver and lung CRC metastasis models, CAC mice models, transmission electron microscopy, immunohistochemistry, histopathology, 4D label-free proteomics sequencing, bioinformatic and database analysis were used in this study. RESULTS: Here, we investigated that MSI2 was upregulated in CRC and positively correlated with ferroptosis inhibitor molecules. MSI2 deficiency suppressed CRC malignancy by inhibiting cell proliferation, viability, migration and invasion in vitro and in vivo; and MSI2 deficiency triggered CRC ferroptosis by changing the intracellular redox state (ROS levels and lipid peroxidation), erastin induced cell mortality and viability, iron homeostasis (intracellular total irons and ferrous irons), reduced glutathione (GSH) levels and mitochondrial injury. Mechanistically, through 4D-lable free proteomics analysis on SW620 stable cell lines, we demonstrated that MSI2 directly interacted with p-ERK and MSI2 knockdown downregulated the p-ERK/p38/MAPK axis signaling pathway, which further repressed MAPKAPK2 and HPSB1 phosphorylation, leading to decreased expression of PCNA and Ki67 and increased expression of ACSL4 in cancer cells. Furthermore, HSPB1 could rescue the phenotypes of MSI2 deficiency on CRC ferroptosis in vitro and in vivo. CONCLUSIONS: This study indicates that MSI2 deficiency suppresses the growth and survival of CRC cells and promotes ferroptosis by inactivating the MAPK signaling pathway to inhibit HSPB1 phosphorylation, which leads to downregulation of PCNA and Ki67 and upregulation of ACSL4 in cancer cells and subsequently induces redox imbalance, iron accumulation and mitochondrial shrinkage, ultimately triggering ferroptosis. Therefore, targeted inhibition of MSI2/MAPK/HSPB1 axis to promote ferroptosis might be a potential treatment strategy for CRC.

Analytical performance evaluation of five RT-PCR kits for severe acute respiratory syndrome coronavirus 2.

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.

Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer.

OBJECTIVE: To evaluate the effect of a new type of automatic glycated hemoglobin analyzer on the separation of abnormal hemoglobin. METHODS: Samples diagnosed as hemoglobin variants by capillary electrophoresis and gene testing were selected, and HbA1c analyzer was used for separation and detection. RESULTS: A total of 13 hemoglobin variants in 40 samples could be separated from the normal peaks. CONCLUSIONS: The variant mode of hemoglobin HbA1c can identify a variety of hemoglobin variants, and the type of variants can be preliminarily determined according to the retention time and characteristic peak shape of the variants.

Consistency evaluation of Liferiver, Yaneng, Darui, and the Cobas 4800 test for high-risk human papillomavirus screening.

BACKGROUND: In recent years, several high-risk human papillomavirus (HR-HPV) tests have been developed. The assay capabilities need to be systematically reviewed. Here, we compared the clinical sample performance of three novel HR-HPV assays (Liferiver, Yaneng, and Darui) based on different platforms with the widely adopted cobas4800 test. METHODS: A total of 346 cervical swabs from women who were screened for cervical cancer were analyzed for the presence of 14 HR-HPV genotypes. The distinction between the four assays was investigated by the genotyping and direct sequencing. RESULTS: The positive rates of the four assays ranged from 61.56% to 64.16%. The overall concordance was 88.15%. The Yaneng assays displayed the best sensitivity (100%) and specificity (98.43%). The sensitivity (98.17%) and specificity (98.43%) of the Darui assay were superior to those of the cobas4800 test (97.72% and 93.70%, respectively). The Liferiver assay displayed comparable sensitivity with the cobas4800 test (95.89% and 97.72%, respectively). The specificity of the cobas4800 was lower than that of the Liferiver assay (93.70% vs. 97.64%). CONCLUSIONS: The three novel HR-HPV assays displayed good agreement with the cobas4800 test. The analytical performance of all four fulfilled the requirements of sensitivity and specificity for HR-HPV detection.

HOXA13 contributes to gastric carcinogenesis through DHRS2 interacting with MDM2 and confers 5-FU resistance by a p53-dependent pathway.

5-FU-based chemotherapy is recently most recommended as the first-line treatment for gastric cancer (GC). However, 5-FU resistance is common for many postoperative GC patients. Homeobox A13 (HOXA13) is a member of homeobox genes highly expressed in many human tumors. Its potential roles and mechanisms of resistance to 5-FU in GC are poorly understood. In this study, we discovered that HOXA13 played an oncogenic role in vivo and in vitro. The patients with HOXA13 overexpression were closely related with poor prognosis and more prone to be resistant to 5-FU. Moreover, dehydrogenase/reductase 2 (DHRS2) was identified as a downstream gene of HOXA13. HOXA13 played a role of carcinogenesis through directly down-regulating DHRS2 to increase MDM2. Furthermore, HOXA13 conferred 5-FU resistance through MRP1 by a p53-dependent pathway. Therefore, HOXA13 might serve as a potential signature that recognized patients who were insensitive to 5-FU, and timely recommended them to other chemotherapy regimens.

Correction to: Metalloproteases meprin-ɑ (MEP1A) is a prognostic biomarker and promotes proliferation and invasion of colorectal cancer.

CORRECTION: After publication of the original article [1] the authors found that the figure contained an incorrect version of Fig. 3a. This does not affect the results and conclusions of the article.

miR-4775 promotes colorectal cancer invasion and metastasis via the Smad7/TGFß-mediated epithelial to mesenchymal transition.

BACKGROUND: Despite advancements in the diagnosis and treatment of colorectal cancer (CRC), many patients die because of tumor metastasis or recurrence. Therefore, identifying new prognostic markers and elucidating the mechanisms of CRC metastasis and recurrence will help to improve the prognosis of the disease. As dysregulation of microRNAs is strongly related to cancer progression, the aim of this study was to identify the role of miR-4775 in the prognosis of CRC patients and the underling mechanisms involved in CRC progression. METHODS: qPCR and in situ hybridization were used to evaluate the expression of miR-4775 in 544 pairs of paraffin-embedded normal and CRC tissues. Kaplan-Meier analysis with the log-rank test was used for survival analyses. Immunohistochemical staining was applied to investigate the expression of miR-4775-regulated Smad7/TGFß pathway-associated markers. In vitro and in vivo invasion and metastasis assays were used to explore the function of miR-4775 in the progression of CRC. RESULTS: miR-4775 was identified as a high-risk factor for CRC metastasis and recurrence, with high levels predicting poor survival among the 544 studied CRC patients. Furthermore, high miR-4775 expression promoted the invasion of CRC cells as well as metastasis and the epithelial to mesenchymal transition (EMT) via Smad7-mediated activation of TGFß signaling both in vitro and in vivo. Downregulating miR-4775 or overexpressing Smad7 reversed the tumor-promoting roles of miR-4775/Smad7/TGFß in vitro and in vivo. CONCLUSION: miR-4775 promotes CRC metastasis and recurrence in a Smad7/TGFß signaling-dependent manner, providing a new therapeutic target for inhibiting the metastasis or recurrence of the disease.

Down-regulation of Barx2 predicts poor survival in colorectal cancer.

Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, has an important role in controlling the expression of cell adhesion molecules and has been reported in an increasing array of tumor types except colorectal cancer (CRC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in CRC. First, we analyzed the expression of Barx2 in two independent public datasets from Oncomine. Subsequently, we evaluated Barx2 mRNA and protein expression by quantitative real-time PCR and western blotting, respectively. It was determined that Barx2 expression was lower in tumor tissues than in adjacent non-tumorous colorectal tissues of CRC patients, consistent with results from the public datasets. Subsequently, a tissue microarray containing 196 CRC specimens was evaluated for Barx2 expression by immunohistochemical staining. It was found that low expression of Barx2 significantly correlated with TNM stage, AJCC stage, differentiation, and relapse in patients with CRC. Patients with lower levels of Barx2 expression showed reduced disease-free survival and overall survival. Furthermore, a trend toward shorter overall survival in the patient group with Barx2-negative tumors independent of advanced AJCC stage and poor differentiation was determined by Kaplan-Meier survival analysis. Based on univariate and multivariate analyses, Barx2 expression was an independent prognostic factor for determining CRC prognosis. Taken together, low Barx2 expression was associated with the progression of CRC and could serve as a potential independent prognostic biomarker for patients with CRC.

Elevated OCT1 participates in colon tumorigenesis and independently predicts poor prognoses of colorectal cancer patients.

Octamer transcription factor 1 (OCT1) was found to influence the genesis and progression of numerous cancers except for colorectal cancer (CRC). This study tried to explore the role of OCT1 in CRC and clarify the association between its expression and patients' clinical outcome. Transcriptional and post-transcriptional expression of OCT1 was detected in CRC cancerous tissues and paired normal mucosae by real-time PCR as well as immunohistochemistry. Moreover, the effect of OCT1 knockdown on CRC cell proliferation was investigated both in vitro and in vivo using Cell Counting Kit-8 assay, colony-forming assay, and mouse tumorigenicity assay. Expression of OCT1 was found to be elevated in CRC. Suppression of OCT1 significantly inhibited CRC cell proliferation both in vitro and in vivo. Furthermore, upregulated level of OCT1 was significantly associated with N stage, M stage, and American Joint Committee on Cancer (AJCC) stage (P = 0.027, 0.014, and 0.002, respectively) as well as differential degree (P = 0.022). By using multivariate Cox hazard model, OCT1 was also shown to be a factor independently predicting overall survival (OS; P = 0.013, hazard ratio = 2.747, 95 % confidence interval 1.125 to 3.715) and disease-free survival (DFS; P = 0.004, hazard ratio = 2.756, 95 % confidence interval 1.191 to 4.589) for CRC patients. Our data indicate that OCT1 carries weight in colorectal carcinogenesis and functions as a novel prognostic indicator and a promising target of anti-cancer therapy for CRC.

Metalloproteases meprin-ɑ (MEP1A) is a prognostic biomarker and promotes proliferation and invasion of colorectal cancer.

BACKGROUND: Meprin displays multiple functions in both health and disease, due in part to its broad proteolytic activity. In this report, we explored the clinical significance and functional relevance of the expression of meprin-ɑ (MEP1A) in colorectal cancer (CRC). METHODS: The mRNA and protein expression levels of MEP1A in tumor specimens obtained from CRC patients was determined by quantitative real-time PCR and Western blot assay and comparatively paired with adjacent mucosa that presented as normal tissue. ShRNA was used to knock-down MEP1A expression in CRC cell-lines and the effects of dampened expression of MEP1A on the proliferation and invasion were determined by colony formation assays, Cell Counting Kit-8 assays and matrigel invasion assays. Moreover, nude mouse xenograft models were designed to investigate the same effect in vivo. In order to determine whether MEP1A expression correlated with CRC clinicopathologic factors and survival, immunohistochemical staining of a tissue microarray containing 88 paired CRC specimens was performed. RESULTS: In CRC, enhanced expression of MEP1A was seen. Additionally, both in vitro and in vivo, CRC cellular proliferation and invasiveness was inhibited by dampened MEP1A expression. Several parameters were associated with enhanced MEP1A expression including tumor size (P = 0.023), staging of CRC by the American Joint Committee on Cancer (AJCC) (P = 0.024), and T (P = 0.032) and N stages (P = 0.001). Moreover, the expression of MEP1A is an independent prognostic factor for overall survival in CRC (HR 3.643; 95 % CI 0.305-5.842; P = 0.007). CONCLUSION: MEP1A was not only found to be functionally important, but it might also serve as an important and unique indicator of patient prognosis and therapeutic targeting in CRC.

Ubiquitin D is an independent prognostic marker for survival in stage IIB-IIC colon cancer patients treated with 5-fluoruracil-based adjuvant chemotherapy.

BACKGROUND: Postoperative 5-fluoruracil (5-FU)-based adjuvant chemotherapy is recommended for stage II colon cancer patients with high conventional risk factors; however, some of these patients still experience tumor recurrence. Identifying novel biomarkers to distinguish the risk of tumor recurrence after surgery is vital for improving their prognoses. We previously showed that ubiquitin D (UBD) can predict the prognosis of colon cancer; however, there are limited data on whether UBD is an independent prognostic factor for stage II patients treated with 5-FU-based adjuvant chemotherapy. METHODS: Quantitative real-time PCR and Western blot analyses were used to examine UBD expression in randomly selected stage II patients' tumor tissues. UBD expression and p65 distribution were assessed using immunohistochemistry in paraffin-embedded specimens from the 101 tumor recurrence patients and 178 nonrelapse patients who received postoperative 5-FU-based adjuvant chemotherapy. RESULTS: UBD expression, both at transcriptional and posttranscriptional levels, was higher in relapse tumors (P < 0.001). Immunohistochemistry staining of UBD and p65 showed significant differences between the two groups (P < 0.001). Patients with tumor tissues that UBD-positive expression alone or in combination with p65 nuclei translocation recurred early had a significantly shorter survival time (P < 0.001), especially in stage IIB-IIC patients. UBD-positive expression accompanied with p65 nuclei translocation was a significant independent predictive high risk factor for overall survival (HR 8.76; 95% CI, 5.35-14.27; P = 0.004) and disease-free survival (HR 5.70; 95% CI, 1.43-11.55; P = 0.016). CONCLUSION: UBD may help to identify recurrent risk in stage IIB-IIC colon cancer patients and further predict which patients benefit from postoperative 5-FU-based adjuvant chemotherapy.

Mapping of hepatic expression quantitative trait loci (eQTLs) in a Han Chinese population.

BACKGROUND: Elucidating the genetic basis underlying hepatic gene expression variability is of importance to understand the aetiology of the disease and variation in drug metabolism. To date, no genome-wide expression quantitative trait loci (eQTLs) analysis has been conducted in the Han Chinese population, the largest ethnic group in the world. METHODS: We performed a genome-wide eQTL mapping in a set of Han Chinese liver tissue samples (n=64). The data were then compared with published eQTL data from a Caucasian population. We then performed correlations between these eQTLs with important pharmacogenes, and genome-wide association study (GWAS) identified single nucleotide polymorphisms (SNPs), in particular those identified in the Asian population. RESULTS: Our analyses identified 1669 significant eQTLs (false discovery rate (FDR) < 0.05). We found that 41% of Asian eQTLs were also eQTLs in Caucasians at the genome-wide significance level (p=10⁻8). Both cis- and trans-eQTLs in the Asian population were also more likely to be eQTLs in Caucasians (p<10⁻4). Enrichment analyses revealed that trait-associated GWAS-SNPs were enriched within the eQTLs identified in our data, so were the GWAS-SNPs specifically identified in Asian populations in a separate analysis (p<0.001 for both). We also found that hepatic expression of very important pharmacogenetic (VIP) genes (n=44) and a manually curated list of major genes involved in pharmacokinetics (n=341) were both more likely to be controlled by eQTLs (p<0.002 for both). CONCLUSIONS: Our study provided, for the first time, a comprehensive hepatic eQTL analysis in a non-European population, further generating valuable data for characterising the genetic basis of human diseases and pharmacogenetic traits.

Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer.

Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.

A novel model for orthotopic liver transplantation in rats using hepatic rearterialization and biliary extradrainage system.

BACKGROUND: Although the rat orthotopic liver transplantation (OLT) model has existed for many years, only a few models can be applied for dynamic bile collection. The aim of this study was to introduce a dependent rat OLT model with hepatic rearterialization and an expediently dynamic bile collection system. METHODS: Forty-five male Sprague-Dawley rats were divided into the following three groups (n = 15 each): group A, OLT without hepatic rearterialization; group B, OLT with hepatic rearterialization; group C, OLT with hepatic rearterialization and a biliary extradrainage system. In groups B and C, a modified sleeve anastomosis between the donor common hepatic artery and the recipient proper hepatic artery was performed to restore the hepatic artery blood flow. In group C, after hepatic rearterialization, biliary extradrainage and jejunum stoma were performed to reestablish the bile flow, and a waistcoat-like external fixator was introduced to protect this system. RESULTS: The surgical success rates in groups A, B, and C were 100% (15/15), 93% (14/15), and 93% (14/15), respectively. In groups B and C, the hepatic artery patency rates were 93% and 86% on postoperative day 3 and postoperative day 21, respectively. Also, the liver function and bile duct integrity were preserved better than that in group A. In group C, the biliary extradrainage system was well preserved and bile collection was easily performed. CONCLUSIONS: The rat OLT model with hepatic rearterialization and a convenient biliary extradrainage system was satisfactory in maintaining the survival rate, hepatic artery patency rate, and recovery of graft function, so it can be applied in various studies after transplantation.

Zinc-α-2-glycoprotein: a candidate biomarker for colon cancer diagnosis in Chinese population.

Zinc-α-2-glycoprotein (AZGP1) is a 41-kDa secreted glycoprotein, which has been detected in several malignancies. The diagnostic value of AZGP1 in serum of prostate and breast cancer patients has been reported. Analyzing "The Cancer Genome Atlas" data, we found that in colon cancer AZGP1 gene expression was upregulated at transcriptional level. We hypothesized that AZGP1 could be used as a diagnostic marker of colon cancer. First, we confirmed AZGP1 expression was higher in a set of 28 tumor tissues than in normal colonic mucosa tissues by real-time quantitative PCR and western blot in a Chinese population. We verified that serum concentration of AZGP1 was higher in 120 colon cancer patients compared with 40 healthy controls by ELISA (p < 0.001). Then receiver-operating characteristic (ROC) curve analysis was used to evaluate the predictive diagnostic value of AZGP1 in serum. The area under the curve (AUC) of AZGP1 was 0.742 (p < 0.001, 95% confidence interval (CI) = 0.656-0.827) in between the AUC of carcinoembryonic antigen (CEA) and the AUC of CA19-9, suggesting that predictive diagnostic value of AZGP1 is between CEA and Carbohydrate 19-9 (CA19-9). The combination of AZGP1 with traditional serum biomarkers, CEA and CA19-9, could result in better diagnostic results. To further validate the diagnostic value of AZGP1, a tissue microarray containing 190 samples of primary colon cancer tissue paired with normal colonic tissue was analysed and the result showed that AZGP1 was significantly upregulated in 68.4% (130 of 190) of the primary cancer lesions. In contrast, there was a weakly positive staining in 29.5% (56 of 190) of the normal colonic tissue samples (p < 0.001). Leave-one-out cross-validation was performed on the serum data, and showed that the diagnostic value of AZGP1 had 63.3% sensitivity and 65.0% specificity. Combination of AZGP1, CEA and CA19-9 had improved diagnosis value accuracy with 74.2% sensitivity and 72.5% specificity. These results suggest that AZGP1 is a useful diagnostic biomarker in tissues and serum from a Chinese population.

Fast, portable and sensitive detection of group B streptococcus DNA using one-pot MIRA-CRISPR system with suboptimal PAM.

The group B Streptococcus (GBS) can generate vertical transmission to infants during delivery, has been seriously threatening the health of infants. Rapid and accurate prenatal GBS diagnosis for pregnant women is a deterministic blueprint to avoid infant viruses. Here, we developed an extraction-free nucleic acid isothermal amplification/CRISPR-Cas12a cutting one-pot system for GBS diagnostic assay by using suboptimal protospacer adjacent motifs, effectively avoiding multiple handling steps and uncapping contamination. The GBS diagnosis assay based on a one-pot system was validated by using fluorescent technique and lateral flow assay strips, exhibited fantastic specificity, accuracy and sensitivity with a limit of detection of 32 copies per reaction (0.64 copies/µL). Moreover, a portable device was constructed and integrated with the one-pot system to realize the GBS detection without professional and scene restrictions, it showed excellent performance in clinical sample detection, which achieved optical and portable GBS detection for point-of-care testing or home-self testing.

Downregulation of metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in colon cancer tissue.

PURPOSE: Downregulation of metallothionein (MT) genes has been reported in several tumors with discrepant results. This study is to investigate molecular mechanism of MT gene regulation in colon cancer which is characterized by tumor suppressor gene alterations. EXPERIMENTAL DESIGN: Integral analysis of microarray data with loss of heterozygosity (LOH) information was employed. Quantitative real-time PCR and immunohistochemistry were used to validate MT isoform expression in colon cancer tissues and cell lines. The effects of MT1F expression on RKO cell survival and tumorigenesis was analyzed. Bisulphite sequencing PCR (BSP) and methylation-specific PCR were employed to detect the methylation status of the MT1F gene in colon cancer tissues and cell lines. DNA sequencing was used to examine the LOH at the MT1F locus. RESULTS: MT1F, MT1G, MT1X, and MT2A gene expression was significantly downregulated in colon cancer tissue (p<0.05). Exogenous MT1F expression increased RKO cell apoptosis and inhibited RKO cell migration, invasion and adhesion as well as in vivo tumorigenicity. Downregulation of MT1F gene in majority of human colon tumor tissues is mainly through mechanism by loss of heterozygosity (p=0.001) while CpG island methylation of MT1F gene promoter region was only observed in poorly differentiated, MSI-positive RKO and LoVo colon cancer cell lines. CONCLUSIONS: MT1F is a putative tumor suppressor gene in colon carcinogenesis that is downregulated mainly by LOH in colon cancer tissue. Further studies are required to elucidate a possible role for MT1F downregulation in colon cancer initiation and/or progression.