Ascitic microbiota alteration is associated with portal vein tumor thrombosis occurrence and prognosis in hepatocellular carcinoma.

Portal vein tumor thrombosis (PVTT) frequently leads to malignant ascites (MA) in individuals with hepatocellular carcinoma (HCC), remaining a bottleneck in the treatment. This study aimed to explore the differences in microbes in paired groups and provide novel insights into PVTT and MA-related treatments. Formalin-fixed paraffin embedding ascite samples were collected from MA secondary to HCC and benign ascites (BA) secondary to liver cirrhosis (LC). Ascitic microbiota profiles were determined in the HCC and LC groups by 16S rRNA sequencing. Prognostic risk factors were screened using survival analysis. The correlation between the significantly different microbial signatures in the groups with PVTT (WVT) and non-PVTT (NVT) and clinical characteristics was explored. The expression of different immune cells was determined by labeling four markers in the MA tissue chips using multiplex immunohistochemistry. A total of 240 patients (196 with HCC with MA and 44 with LC with BA) were included in this study. Microbial profiles differed between the HCC and LC groups. PVTT and Barcelona Clinic Liver Cancer stage were shown to be prognostic risk factors. Significant differences in the alpha and beta diversities were observed between the WVT and NVT groups. Gammaproteobacteria and Acinetobacter were the most abundant in the HCC MA. Differences in microbial signatures between the WVT and NVT groups were correlated with the level of C-reactive protein and apolipoprotein A1. This study revealed the microbial differences in the tumor microenvironment of MA secondary to HCC and BA secondary to LC.IMPORTANCEFirst, we explored the alteration of the ascites ecosystem through the microbiota in patients with hepatocellular carcinoma (HCC) and liver cirrhosis. Second, this is the first clinical study to investigate the differences between patients with HCC with and without portal vein tumor thrombosis via 16S rRNA sequencing. These results revealed a decreased microbial diversity and metabolic dysregulation in individuals with HCC and portal vein tumor thrombosis. Gammaproteobacteria and Acinetobacter were the most abundant in the HCC malignant ascitic fluid. Our study provides a new perspective on treating malignant ascites secondary to HCC.

Association between ascites Gustave Roussy immune score and the intratumoural microbiome in malignant ascites secondary to hepatocellular carcinoma.

BACKGROUNDS: The Gustave Roussy Immune (GRIm) score predicts survival outcomes in several cancers. However, the prognostic significance of the GRIm score in patients with malignant ascites has not yet been investigated. METHODS: Clinical samples were collected from a cohort of patients with malignant ascites secondary to hepatocellular carcinoma (HCC). We calculated serum GRIm (sGRIm) and ascites GRIm (aGRIm) scores and divided the samples into low and high GRIm score groups. Survival analysis was used to compare the prognostic significance of the sGRIm and aGRIm scores. 16S rRNA sequencing was performed to determine the profiles of the intratumoral microbiota in the groups. A fluorescent multiplex immunohistochemistry (mIHC) assay was used to detect the expression of different immune cells by labeling seven markers of malignant ascites. RESULTS: 155 patients with HCC and malignant ascites were enrolled in this study. Survival analysis revealed that the aGRIm score showed a superior prognostic significance compared to the sGRIm score. Microbial analysis demonstrated that the bacterial richness and diversity were higher in the low aGRIm score group than in the high aGRIm score group. LefSe analysis revealed that certain bacteria were correlated with high aGRIm scores. Fluorescent mIHC displayed the tumor microenvironment of malignant ascites and found that the density of CD8 + T cells was significantly higher in the low aGRIm score group than in the high aGRIm score group. CONCLUSIONS: Our present study identified a novel scoring system (aGRIm score) that can predict the survival outcome of patients with malignant ascites secondary to HCC.

The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

Kinome-Wide Virtual Screening by Multi-Task Deep Learning.

Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.

Pterostilbene suppresses gastric cancer proliferation and metastasis by inhibiting oncogenic JAK2/STAT3 signaling: In vitro and in vivo therapeutic intervention.

BACKGROUND: Gastric cancer (GC) represents a significant health burden with dire prognostic implications upon metastasis and recurrence. Pterostilbene (PTE) has been proven to have a strong ability to inhibit proliferation and metastasis in other cancers, while whether PTE exhibits anti-GC activity and its potential mechanism remain unclear. PURPOSE: To explore the efficacy and potential mechanism of PTE in treating GC. METHODS: We employed a comprehensive set of assays, including CCK-8, EdU staining, colony formation, flow cytometry, cell migration, and invasion assays, to detect the effect of PTE on the biological function of GC cells in vitro. The xenograft tumor model was established to evaluate the in vivo anti-GC activity of PTE. Network pharmacology was employed to predict PTE's potential targets and pathways within GC. Subsequently, Western blotting, immunofluorescence, and immunohistochemistry were utilized to analyze protein levels related to the cell cycle, EMT, and the JAK2/STAT3 pathway. RESULTS: Our study demonstrated strong inhibitory effects of PTE on GC cells both in vitro and in vivo. In vitro, PTE significantly induced cell cycle arrest at G0/G1 and S phases and suppressed proliferation, migration, and invasion of GC cells. In vivo, PTE led to a dose-dependent reduction in tumor volume and weight. Importantly, PTE exhibited notable safety, leaving mouse weight, liver function, and kidney function unaffected. The involvement of the JAK2/STAT3 pathway in PTE's anti-GC effect was predicted utilizing network pharmacology. PTE suppressed JAK2 kinase activity by binding to the JH1 kinase structural domain and inhibited the downstream STAT3 signaling pathway. Western blotting confirmed PTE's inhibition of the JAK2/STAT3 pathway and EMT-associated protein levels. The anti-GC effect was partially reversed upon STAT3 activation, validating the pivotal role of the JAK2/STAT3 signaling pathway in PTE's activity. CONCLUSION: Our investigation validates the potent inhibitory effects of PTE on the proliferation and metastasis of GC cells. Importantly, we present novel evidence implicating the JAK2/STAT3 pathway as the key mechanism through which PTE exerts its anti-GC activity. These findings not only establish the basis for considering PTE as a promising lead compound for GC therapeutics but also contribute significantly to our comprehension of the intricate molecular mechanisms underlying its exceptional anti-cancer properties.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment.

OBJECTIVE: The correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and degradation of bile metabolites. DESIGN: Differential metabolites in the bile from CCA and benign biliary stenosis were identified by metabonomics. Biliary molecules able to induce mast cell (MC) degranulation were revealed by in vitro and in vivo experiments, including liquid chromatography-mass spectrometry (MS)/MS and bioluminescence resonance energy transfer assays. Histamine (HA) receptor expression in CCA was mapped using a single-cell mRNA sequence. HA receptor functions were elucidated by patient-derived xenografts (PDX) in humanised mice and orthotopic models in MC-deficient mice. Genes involved in HA-induced proliferation were screened by CRISPR/Cas9. RESULTS: Bile HA was elevated in CCA and indicated poorer prognoses. Cancer-associated fibroblasts (CAFs)-derived stem cell factor (SCF) recruited MCs, and bile N,N-dimethyl-1,4-phenylenediamine (DMPD) stimulated MCs to release HA through G protein-coupled receptor subtype 2 (MRGPRX2)-Gαq signalling. Bile-induced MCs released platelet-derived growth factor subunit B (PDGF-B) and angiopoietin 1/2 (ANGPT1/2), which enhanced CCA angiogenesis and lymphangiogenesis. Histamine receptor H1 (HRH1) and HRH2 were predominantly expressed in CCA cells and CAFs, respectively. HA promoted CCA cell proliferation by activating HRH1-Gαq signalling and hastened CAFs to secrete hepatocyte growth factor by stimulating HRH2-Gαs signalling. Solute carrier family 22 member 3 (SLC22A3) inhibited HA-induced CCA proliferation by importing bile HA into cells for degradation, and SLC22A3 deletion resulted in HA accumulation. CONCLUSION: Bile HA is released from MCs through DMPD stimulation and degraded via SLC22A3 import. Different HA receptors exhibit a distinct expression profile in CCA and produce different oncogenic effects. MCs promote CCA progression in a CCA-bile interplay pattern.

Polydopamine-assisted aptamer-carrying tetrahedral DNA microelectrode sensor for ultrasensitive electrochemical detection of exosomes.

BACKGROUND: Exosomes are nanoscale extracellular vesicles (30-160 nm) with endosome origin secreted by almost all types of cells, which are considered to be messengers of intercellular communication. Cancerous exosomes serve as a rich source of biomarkers for monitoring changes in cancer-related physiological status, because they carry a large number of biological macromolecules derived from parental tumors. The ultrasensitive quantification of trace amounts of cancerous exosomes is highly valuable for non-invasive early cancer diagnosis, yet it remains challenging. Herein, we developed an aptamer-carrying tetrahedral DNA (Apt-TDNA) microelectrode sensor, assisted by a polydopamine (PDA) coating with semiconducting properties, for the ultrasensitive electrochemical detection of cancer-derived exosomes. RESULTS: The stable rigid structure and orientation of Apt-TDNA ensured efficient capture of suspended exosomes. Without PDA coating signal amplification strategy, the sensor has a linear working range of 102-107 particles mL-1, with LOD of ~ 69 exosomes and ~ 42 exosomes for EIS and DPV, respectively. With PDA coating, the electrochemical signal of the microelectrode is further amplified, achieving single particle level sensitivity (~ 14 exosomes by EIS and ~ 6 exosomes by DPV). CONCLUSIONS: The proposed PDA-assisted Apt-TDNA microelectrode sensor, which integrates efficient exosome capture, sensitive electrochemical signal feedback with PDA coating signal amplification, provides a new avenue for the development of simple and sensitive electrochemical sensing techniques in non-invasive cancer diagnosis and monitoring treatment response.

A dual amplification-based CRISPR/Cas12a biosensor for sensitive detection of miRNA in prostate cancer.

MicroRNA (miRNA) has gained significant attention as a potential biomarker for cancer clinics, and there is an urgent need for developing sensing strategies with high selectivity, sensitivity, and low background. In vitro diagnosis based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated protein (CRISPR/Cas) technology could simplify the detection procedure, improve sensitivity and selectivity, and has broad application prospects as the next-generation molecular diagnosis technology. We propose a novel dual signal amplification strategy, called CENTER, which integrates the CRISPR/Cas12a system, an entropy-driven DNA signaling network, and strand displacement amplification to achieve ultrasensitive detection of miR-141, a potential marker for prostate cancer. The experimental results demonstrate that CENTER can distinguish single nucleotide mutations, and the strategy exhibits a good linear calibration curve ranging from 100 aM to 1 pM. Due to dual signal amplification, the detection limit is as low as 34 aM. We proposed a method for identifying miR-141 expressed in human serum and successfully distinguished between prostate cancer patients (n = 20) and healthy individuals (n = 15) with an impressive accuracy of 94%. Overall, CENTER shows great promise for the detection of miRNA.

The value of serum HE4 and CA125 levels for monitoring the recurrence and risk stratification of endometrial endometrioid carcinoma.

To evaluate the role of serum human epididymis secretory protein 4 (HE4) and carbohydrate antigen 125 (CA125) levels for predicting and monitoring the recurrence of endometrial endometrioid carcinoma (EEC) and assessing preoperative risk stratification in EEC patients. A total of 434 EEC patients were selected for this retrospective study between May 2011 and August 2018. Serum HE4 and CA125 levels were analyzed before the initial treatment, at the first postoperative follow-up, and at recurrence or the last follow-up. Patients were risk stratified according to the European Society for Medical Oncology (ESMO), European Society for Radiotherapy & Oncology (ESTRO) and European Society of Gynaecological Oncology (ESGO) guideline. We compared the ability of these biomarkers for prediction and monitoring by performing receiver operating characteristic curve analysis and identified optimal cut-off values by determining the Youden index. Kaplan-Meier analyses were also performed to determine prognostic value. Preoperative serum HE4 was identified as a significant predictor for the recurrence of EEC (p = 0.014). Preoperative serum HE4 and CA125 levels were related to depth of myometrial invasion, lymph node status and FIGO stage. Serum HE4 and CA125 levels were both statistically significant markers for monitoring the recurrence of EEC (P = 0.000 for each biomarker). When combined, the two markers showed higher levels of sensitivity and specificity. The two biomarkers were also significant biomarkers for evaluating the risk stratification of patients undergoing lymphadenectomy (P = 0.000 for each biomarker). For premenopausal stage I patients, preoperative serum HE4 and CA125 levels were significant predictors of the need for ovarian preservation (P = 0.000 and P = 0.002, respectively). For premenopausal patients with stage I intramucosal differentiation, preoperative serum levels of HE4 were significant predictors for fertility preservation (P = 0.024). Preoperative serum HE4 level can be used to predict the recurrence of EEC. Postoperative serum HE4 and CA125 levels can be used to monitor the recurrence of EEC and are more sensitive when combined. Preoperative serum levels of CA125 and HE4 levels are of significant value for risk stratification in EEC patients.

Higher clearance rates of multiple HPV infections may explain their lower risk of HSIL: A retrospective study in Wenzhou, China.

Persistent human papilloma virus (HPV) infection is known to be associated with cervical lesions. The chief object of the study is to investigate if the pathogenicity of multiple HPV infections is different from a single infection. Furthermore, we would like to corroborate the discrepancy with clearance rates. Between August 1, 2020, and September 31, 2021, 5089 women underwent a colposcopy-directed biopsy in our hospital. We divided the 2999 patients who met the criteria into multiple and single HPV infection groups. The HPV genotypes were identified using the flow cytometry fluorescence hybridization technology. Binary logistic regression and survival analysis were used to perform statistics. Among HPV-positive individuals, 34.78% (1043/2999) were positive for 2 or more HPV types. After adjusting for the main factors, compared with single infection, multiple infections were associated with a significantly decreased risk of high squamous intraepithelial lesions (HSIL) (odds ratio [OR]: 0.570; 95% confidence interval [CI]: 0.468-0.694). In the mean time, the clearance rates of multiple infections were significantly higher (OR: 2.240; 95% CI: 1.919-2.614). When analyzing specific types covered by the 9-valent HPV vaccine, consistency between the lower risk of HSIL and the higher clearance rate was found in the most groups. Compared with a single infection, multiple HPV infections have a lower risk of HSIL, which may be related to its higher clearance rate. It suggests that aggressive treatment of multiple HPV infections early in their detection may be beneficial.

Analysis and Biomedical Applications of Functional Cargo in Extracellular Vesicles.

Extracellular vesicles (EVs) can facilitate essential communication among cells in a range of pathophysiological conditions including cancer metastasis and progression, immune regulation, and neuronal communication. EVs are membrane-enclosed vesicles generated through endocytic origin and contain many cellular components, including proteins, lipids, nucleic acids, and metabolites. Over the past few years, the intravesicular content of EVs has proven to be a valuable biomarker for disease diagnostics, involving cancer, cardiovascular diseases, and central nervous system diseases. This review aims to provide insight into EV biogenesis, composition, function, and isolation, present a comprehensive overview of emerging techniques for EV cargo analysis, highlighting their major technical features and limitations, and summarize the potential role of EV cargos as biomarkers in disease diagnostics. Further, progress and remaining challenges will be discussed for clinical diagnostic outlooks.

Genital HPV Prevalence, Follow-Up and Persistence in Males and HPV Concordance Between Heterosexual Couples in Wenzhou, China.

Purpose: This study aimed to investigate male HPV infection, re-examination rate, clearance rate and relevant influencing factors as well as HPV infection between heterosexual partners in Wenzhou, China. Methods: The study enrolled 2359 men who accepted ≥1 HPV detection in the First Affiliated Hospital of Wenzhou Medical University between June 2014 and June 2020. An outpatient follow-up was carried out for collecting HPV re-test results among males who were tested HPV positive. In addition, we collected female sexual partners' HPV infection state through outpatient information system or telephone call. For males who had not re-tested HPV more than half a year after the first HPV positive result, a telephone follow-up would be provided. Results: Male HPV prevalence was 39.39% (928/2359) at baseline, of which the median age was 38.00 years. The percentage of co-infection and HR HPV infection was 35.24% (327/928) and 61.08% (874/1431). Genotype-specific concordance of HPV infection among heterosexual partners was 61.19% (123/201). The most common types were HPV6, HPV52, HPV16, HPV53, HPV11, HPV43 and HPV58. Percentage of males finishing HPV re-examination was low (40.09%). Infection with HR HPV, discomfort or HPV-related diseases and sexual partners with HPV infection were possible motivator for male HPV re-test. The medium time to male HPV clearance was 300.000 (95% CI=274.845-325.155) days. Infection with HR HPV, sexual partners with HPV infection and growth of age might prolong HPV persistence time included. Conclusion: Males are susceptible population for genital HPV infection as well as females. Whereas, males are generally less aware of HPV and its influence on their couples. The study showed that male HPV testing as well as the role of males in the diagnosis and treatment of HPV-associated cervical diseases for females should be popularized and co-treatment of couples was necessary.

Patient-directed vs. fixed-volume PEG for colonoscopy preparation: a randomized controlled trial.

Background: Individualization using different volumes of polyethylene glycol is widely regarded as the optimal solution for bowel preparation, while the patient-directed regimen we propose may serve as a reliable individual solution. This study aimed to assess the efficacy, safety, and satisfaction of bowel preparation with a patient-directed regimen. Methods: Patients in the fixed-volume group ingested the same amount of PEG, while those in patient-directed group ingested different amount according to stool consistency or stool water content. Results: After filtering by exclusion criteria, 428 individuals in the fixed-volume group and 103 in the patient-directed group were successfully enrolled and analyzed. Eighty-three (80.6%) individuals in the patient-directed group had a reduced polyethylene glycol volume. There was no significant difference in the bowel preparation efficacy between the two groups (90.0% vs. 90.3%, χ² = 0.01; p = 0.918). Patients in the patient-directed group complained of fewer adverse effects (53.0% vs. 36.9%, χ² = 8.655; p = 0.003), especially vomiting (13.6% vs. 1.0%, χ² = 13.304; p < 0.001). Regarding comfort during bowel preparation, the degree of comfort was not significantly different between groups. Furthermore, the willingness rate for further colonoscopy in the patient-directed group was significantly higher than that in the fixed-volume group (90.3% vs. 77.1%, χ² = 8.912; p < 0.05). Multivariable logistic regression analysis showed that the body mass index served as an independent factor impacting quality of bowel preparation with the patient-directed regimen (OR 1.16, 95% CI 1.00-1.34; p = 0.043). Conclusions: Without decreasing the bowel preparation efficacy, the patient-directed regimen increased the safety and satisfaction of bowel preparation and is expected to be a regular and individual solution for bowel preparation. Individuals with a lower body mass index are more likely to undertake this new regimen. Trial registration number: ChiCTR1900022072 at ChiClinicalTrials.gov.

Accurate diagnosis and prognosis prediction of gastric cancer using deep learning on digital pathological images: A retrospective multicentre study.

BACKGROUND: To reduce the high incidence and mortality of gastric cancer (GC), we aimed to develop deep learning-based models to assist in predicting the diagnosis and overall survival (OS) of GC patients using pathological images. METHODS: 2333 hematoxylin and eosin-stained pathological pictures of 1037 GC patients were collected from two cohorts to develop our algorithms, Renmin Hospital of Wuhan University (RHWU) and the Cancer Genome Atlas (TCGA). Additionally, we gained 175 digital pictures of 91 GC patients from National Human Genetic Resources Sharing Service Platform (NHGRP), served as the independent external validation set. Two models were developed using artificial intelligence (AI), one named GastroMIL for diagnosing GC, and the other named MIL-GC for predicting outcome of GC. FINDINGS: The discriminatory power of GastroMIL achieved accuracy 0.920 in the external validation set, superior to that of the junior pathologist and comparable to that of expert pathologists. In the prognostic model, C-indices for survival prediction of internal and external validation sets were 0.671 and 0.657, respectively. Moreover, the risk score output by MIL-GC in the external validation set was proved to be a strong predictor of OS both in the univariate (HR = 2.414, P < 0.0001) and multivariable (HR = 1.803, P = 0.043) analyses. The predicting process is available at an online website (https://baigao.github.io/Pathologic-Prognostic-Analysis/). INTERPRETATION: Our study developed AI models and contributed to predicting precise diagnosis and prognosis of GC patients, which will offer assistance to choose appropriate treatment to improve the survival status of GC patients. FUNDING: Not applicable.

Single Living Cell Analysis Nanoplatform for High-Throughput Interrogation of Gene Mutation and Cellular Behavior.

The genetic heterogeneities in cancer cells pose challenges to achieving precise drug treatment in a widely applicable manner. Most single-cell gene analysis methods rely on cell lysis for gene extraction and identification, showing limited capacity to provide the correlation of genetic properties and real-time cellular behaviors. Here, we report a single living cell analysis nanoplatform that enables interrogating gene properties and drug resistance in millions of single cells. We designed a Domino-probe to identify intracellular target RNAs while releasing 10-fold amplified fluorescence signals. An on-chip addressable microwell-nanopore array was developed for enhanced electro-delivery of the Domino-probe and in situ observation of cell behaviors. The proof-of-concept of the system was validated in primary lung cancer cell samples, revealing the positive-correlation of the ratio of EGFR mutant cells with their drug susceptibilities. This platform provides a high-throughput yet precise tool for exploring the relationship between intracellular genes and cell behaviors at the single-cell level.

A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 µL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.

[Comparative study on infection rate of different adeno-associated virus for knee joint cartilage in mice].

OBJECTIVE: To explore infection rate of different adeno-associated virus (AAV) on knee joint cartilage in mice and to find a good gene editing tool for mice chondrocytes of knee joint. METHODS: Forty-five 4-week-old SPF C57BL/6 weighed(14.3±0.2) g were selected. According to different injections(6 µl) for right knee joint, mice were divided into 9 different groups, 5 mice in each group. The groups were such as following:control group (normal saline), Vigene 2 group (AAV2 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 5 group (AAV5 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 6 group (AAV6 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 7 group (AAV7 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 8 group (AAV8 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 9 group (AAV9 from vigene biosciences, titer for 1×10¹³ vg/ml), Hanbio DJ group(AAV2-DJ from Hanbio, titer for 1×10¹² vg/ml), Hanbio 5 group (AAV5 from Hanbio, titer for 1×10¹² vg/ml). All AAVs were over-expressed green fluorescent protein(GFP). Knee joint specimens were taken and observed injury of cartilage under stereomicroscope at 30 days after injection, then 10 µm thick frozen sections were prepared. Distribution of green fluorescent protein of meniscus and cartilage of knee joint was observed under fluorescence microscope. RESULTS: Stereomicroscope observation indicated that no obvious lesion was observed in knee joint cartilage of mice after intra-articular injection of AAV. According to frozen sections of knee joints, strong green fluorescence was observed in knee joint cartilage in all AAV experimental groups. Compared with other groups, significantly stronger green fluorescence were observed both in AAV2 and AAV7 groups, whose average fluorescence density was 0.077±0.020 and 0.061±0.022. There were significant differences between two groups and other groups. CONCLUSIONS: AAV could infect chondrocyte of knee joint in vivo by injecting into knee joint cavity. Higher infection efficiency of AAV2 and AAV7 on knee joint cartilage were observed. Local injection of AAV into knee joint cavity could be used as an effective tool for gene editing of knee joint chondrocyte.

Stimuli-responsive Carriers for Controlled Intracellular Drug Release.

BACKGROUND: Stimuli-responsive carriers are a class of drug delivery systems which can change their physicochemical properties and/or structural conformations in response to specific stimuli. Although passive and active drug targeting has proved to reduce the side effects to normal cells, controlled intracellular drug release should be included in drug carriers to enhance the bioavailability of drugs at the disease site. METHODS: This review focuses on several recent advances in the development of stimuli-responsive carriers for spatially and temporally controlled release of therapeutic agents in response to intracellular stimuli, such as pH, redox potential, reactive oxygen species, enzyme and temperature. RESULTS: Among the different types of stimuli, pH-responsive carriers have been mostly used to design intracellular controlled release system. The sharp difference of redox potential between inside and outside cells is attributed to the high variation in concentration of glutathione. ROS-responsive carriers are gaining much attention for selective release of therapeutic agents by sensing oxidative conditions at different levels. The advantages of utilizing enzymes as the trigger of stimuli-responsive carriers include diverse types of enzymes, high selectivity of enzyme catalyzed reactions and the mild reaction conditions involved. Abnormal temperature is another unique stimulus and has been widely used to trigger controlled release of drug in tumor cells. CONCLUSION: Recent developments highlighted in this paper demonstrate that stimuli-responsive carriers possess great potential as a new platform for controlled intracellular drug release.

Overhang molecular beacons encapsulated in tethered cationic lipoplex nanoparticles for detection of single-point mutation in extracellular vesicle-associated RNAs.

Detection of specific extracellular RNAs has been developed for non-invasive cancer diagnosis. However, accurate and efficient identification of RNAs with single-point mutation in cancer cells-derived extracellular vesicles (EVs) is challenging. Herein, we present a unique overhang molecular beacon with internal dye (Ohi-MB) with a stable hairpin structure, fast hybridization kinetics and single mismatch specificity. Ohi-MBs are encapsulated in cationic lipoplex nanoparticles (CLNs) that are tethered on a gold coated glass slide as a chip, which can capture circulating EVs and detect encapsulated target RNAs in-situ in a single step. The capability of detection of single-point mutation by CLN-Ohi-MB is demonstrated in artificial EVs and cancer cells. This CLN-Ohi-MB biochip could quantify single-point mutations in KRAS mRNA (G12C, G12D, G12V) in pancreatic cancer cell-derived EVs and single-point mutations in EGFR mRNA (L858R and T790M) in lung cancer cell-derived EVs with high specificity, not achievable by conventional molecular probes. We show that CLN-Ohi-MB biochip could selectively and sensitively identify single-point mutations in KRAS mRNA in human serum EVs, distinguishing pancreatic cancer patients with different mutations.

A signal-amplifiable biochip quantifies extracellular vesicle-associated RNAs for early cancer detection.

Detection of extracellular vesicle (EV)-associated RNAs with low expression levels in early-stage cancer remains a challenge and is highly valuable. Here, we report a nanoparticle-based biochip that could capture circulating EVs without isolation, brighten encapsulated RNAs, and amplify fluorescence signals in situ in a single step. We confine catalyzed hairpin DNA circuit (CHDC) in cationic lipid-polymer hybrid nanoparticles (LPHNs) that are tethered on a chip. LPHN features a core-shell-corona structure that facilitates the transfer and mixing of CHDC with EV-associated RNAs when forming the LPHN-EV nanocomplex. CHDC is triggered upon target RNA binding and quickly generate amplified signals. The signal amplification efficiency of LPHN-CHDC is demonstrated in artificial EVs, cancer cells, and cancer cell-derived EVs. We show that LPHN-CHDC biochip with signal amplification capability could selectively and sensitively identify low expression glypican-1 mRNA in serum EVs, distinguishing patients with early- and late-stage pancreatic cancer from healthy donors and patients with benign pancreatic disease.