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P53 protein tumor suppressor gene plays a guiding role in the treatment and prognosis of colorectal cancer (CRC). This paper aimed at proposing a feature selection method based on variable clustering to improve positive and negative discrimination of P53 protein in CRC patients. In this approach, we cluster the preprocessed dataset with variables, and then find the features with the largest information value (IV) for each cluster to form a feature subset. We call this method as IV_Cluster. In the actual medical data test, compared with the information value feature selection method, the accuracy of the 10-fold cross-validation logistic regression model increased by 4.4%, 2.0%, and 5.8%, and Kappa values increased by 21.8%, 8.6%, and 22.4%, respectively, under 5, 10, and 15 feature sets.

Escherichia coli (E. coli) O157:H7 is a major foodborne and waterborne pathogen that can threaten human health. Due to its high toxicity at low concentrations, it is crucial to establish a time-saving and highly sensitive in situ detection method. Herein, we developed a rapid, ultrasensitive, and visualized method for detecting E. coli O157:H7 based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. The CRISPR/Cas12a-based system was pre-amplified using the RAA method, which showed high sensitivity and enabled detecting as low as ~1 CFU/mL (fluorescence method) and 1 × 102 CFU/mL (lateral flow assay) of E. coli O157:H7, which was much lower than the detection limit of the traditional real-time PCR technology (103 CFU/mL) and ELISA (104~107 CFU/mL). In addition, we demonstrated that this method still has good applicability in practical samples by simulating the detection in real milk and drinking water samples. Importantly, our RAA-CRISPR/Cas12a detection system could complete the overall process (including extraction, amplification, and detection) within 55 min under optimized conditions, which is faster than most other reported sensors, which take several hours to several days. The signal readout could also be visualized by fluorescence generated with a handheld UV lamp or a naked-eye-detected lateral flow assay depending on the DNA reporters used. Because of the advantages of being fast, having high sensitivity, and not requiring sophisticated equipment, this method has a promising application prospect for in situ detection of trace amounts of pathogens.

Semiconducting polymer dots (Pdots) are novel fluorescent probes with relatively high brightness, fast emission rate, and excellent photostability. This review systematically summarizes their applications for in vitro biosensing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 10

Aim: To investigate the clinical value of tumor markers in extrapleural tumor metastasis assessment of newly diagnosed lung cancer patients. Materials & methods: This study retrospectively analyzed 306 patients diagnosed with lung cancer accompanied by tumor metastasis. Patients were grouped into extrapleural tumor metastasis and intrapleural tumor metastasis. Seven serum tumor markers were included for analysis. Results: The area under curves of receiver operating characteristic curve based on binning decision tree algorithm were above 0.8 in both training and validation sets. A scorecard with a score below 3 suggested extrapleural tumor metastasis in newly diagnosed lung cancer patients. Conclusion: The serum tumor marker-derived model is a convenient and fast approach for extrapleural cavity metastasis assessment, which may provide positive implications in newly diagnosed lung cancer patients.

Learning an effective object detector with little supervision is an essential but challenging problem in computer vision applications. In this paper, we consider the problem of learning a deep convolutional neural network (CNN) based object detector using weakly-supervised and semi-supervised information in the framework of fast region-based CNN (Fast R-CNN). The target is to obtain an object detector as accurate as the fully-supervised Fast R-CNN, but it requires less image annotation effort. To solve this problem, we use weakly-supervised training images (i.e., only the image-level annotation is given) and a few proportions of fully-supervised training images (i.e., the bounding box level annotation is given), that is a weakly- and semi-supervised (WASS) object detection setting. The proposed solution is termed as WASS R-CNN, in which there are two main components. At first, a weakly-supervised R-CNN is firstly trained; after that semi-supervised data are used for finetuning the weakly-supervised detector. We perform object detection experiments on the PASCAL VOC 2007 dataset. The proposed WASS R-CNN achieves more than 85% of a fully-supervised Fast R-CNN’s performance (measured using mean average precision) with only 10% of fully-supervised annotations together with weak supervision for all training images. The results show that the proposed learning framework can significantly reduce the labeling efforts for obtaining reliable object detectors.

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100 molecules/μL showed a strong correlation (R2 = 0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100 molecules/μL showed a strong correlation (R

Background Assessment of colorectal cancer (CRC) lymph node metastasis (LNM) is critical to the decision of surgery, prognosis, and therapy strategy. In this study, we aimed to develop and validate a multiple tumor marker nomogram for predicting LNM in CRC patients. Methods A total of 674 patients who met the inclusion criteria were collected and randomly divided into primary cohort and internal test cohort at a ratio of 7:3. An external test cohort enrolled 178 CRC patients from the West China Hospital. Clinicopathologic variables were obtained from electronic medical records. The least absolute shrinkage and selection operator (LASSO) and interquartile range analysis were carried out for variable dimensionality reduction and feature selection. Multivariate logistic regression analysis was conducted to develop predictive models of LNM. The performance of the established models was evaluated by the receiver operating characteristic (ROC) curve, calibration belt, and clinical usefulness. Results Based on minimum criteria, 18 potential features were reduced to six predictors by LASSO and interquartile range in the primary cohort. The model demonstrated good discrimination and ROC curve (AUC = 0.721 in the internal test cohort, AUC = 0.758 in the external test cohort) in LNM assessment. Good calibration was shown for the probability of CRC LNM in the internal and external test cohorts. Decision curve analysis illustrated that multi-tumor markers nomogram was clinically useful. Conclusions The study proposed a reliable nomogram that could be efficiently and conveniently utilized to facilitate the assessment of individually-tailored LNM in patients with CRC, complementing imaging and biopsy tests.

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. At present, large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 (L1) and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved systematic chromosome rearrangement (SCR) in human cells. The copy number variations (CNVs) of the SCR genomes showed typical patterns that observed in tumor genomes. For example, the most frequent deleted region Chr9p21 containing p15 and p16 tumor suppressor, and the amplified region Chr8q24 containing MYC in tumors were all identified in both SCR cells. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by the SCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival under the severe stress induced by the large-scale chromosomal breaks. In addition, we found that the cells acquired CRISPR-Cas9 resistance after SCR by interfering with the Cas9 mRNA. Our study provided a new application of CRISPR-Cas9 and a practical approach for SCR in complex mammalian genomes.HighlightsRepetitive retroelements with large copy numbers were targeted using CRISPR-Cas9 in human cells.Cells survived after their chromosomal DNAs were heavily cleaved and systematic chromosome rearrangement was achieved.Systematic chromosome rearrangement reshaped the genome and transcriptome of the cells.

The ability to sort and dispense droplets accurately is essential to droplet-based single-cell analysis. Here, we describe a fluorescence-activated single-droplet dispenser (FASD) that is analogous to a conventional fluorescence-activated cell sorter, but sorts droplets containing single cells within an oil emulsion. The FASD system uses cytometric detection and electrohydrodynamic actuation-based single-droplet manipulation, allowing droplet isolation and dispensing with high efficiency and accuracy. The system is compatible with multiwell plates and can be integrated with existing microfluidic devices and large-scale screening systems. By enabling sorting based on single-cell reactions such as PCR, this platform will help expand the basis of cell sorting from mainly protein biomarkers to nucleic acid sequences and secreted biomolecules.

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.

Hepatitis B virus (HBV) infection is one of the global healthcare burdens, and its early diagnosis is crucial for the prevention of HBV-induced chronic hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although different detection approaches have been reported, most of these methods either rely on sophisticated machines or procedures, which limit their use particularly in the high endemic, developing countries. In this work, we report a dual-sensing nanoplatform built on noble metal-molybdenum disulfide (MoS2) nanohybrids, and this platform can detect the HBV DNA target through either fluorometric or colorimetric readouts. The design with the silver nanocluster (AgNC)–MoS2 nanohybrid enables multiplex fluorescent detection, while the HBV DNA-regulated growth of platinum nanoparticles (PtNPs) on the MoS2 nanosheets offers signal-on colorimetric detection. Both AgNC–MoS2 and PtNP–MoS2 nanohybrids show high sensitivity with pico-molar detection limit and single nucleotide specificity, even with the spiked human serum. Collectively, the proposed nanohybrids possess their potential in the use of early HBV diagnosis, particularly suitable for the high endemic areas with limited medical and instrumental supports.

Understanding the variation in community composition and species abundances, i.e., β-diversity, is at the heart of community ecology. A common approach to examine β-diversity is to evaluate directional turnover in community composition by measuring the decay in the similarity among pairs of communities along spatial or environmental distances. We provide the first global synthesis of taxonomic and functional distance decay along spatial and environmental distance by analysing 149 datasets comprising different types of organisms and environments. We modelled an exponential distance decay for each dataset using generalized linear models and extracted r2 and slope to analyse the strength and the rate of the decay. We studied whether taxonomic or functional similarity has stronger decay across the spatial and environmental distances. We also unveiled the factors driving the rate of decay across the datasets, including latitude, spatial extent, realm, and organismal features. Taxonomic distance decay was stronger along spatial and environmental distances compared with functional distance decay. The rate of taxonomic spatial distance decay was the fastest in the datasets from mid-latitudes while the rate of functional decay increased with latitude. Overall, datasets covering larger spatial extents showed a lower rate of decay along spatial distances but a higher rate of decay along environmental distances. Marine ecosystems had the slowest rate of decay. This synthesis is an important step towards a more holistic understanding of patterns and drivers of taxonomic and functional β-diversity.

Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive. A rapid, low-cost HPV detection method is needed for cervical cancer screening in low-resource settings. We previously developed a digital loop-mediated isothermal amplification (dLAMP) assay for rapid, quantitative detection of nucleic acids without the need for thermocycling. This assay employs a microfluidic self-digitization chip to automatically digitize a sample into an array of nanoliter wells in a simple assay format. Here we evaluate the dLAMP assay and self-digitization chip for detection of the commonly tested 14 high-risk HPVs in clinical samples. The dLAMP platform provided reliable genotyping and quantitative detection of the 14 high-risk HPVs with high sensitivity, demonstrating its potential for simple, rapid, and low-cost diagnosis of HPV infection.

ABSTRACT: Early and accurate diagnosis of viruses is critical for control of the pandemic. CRISPR/Cas-based detection of nucleic acid is an emerging technology for molecular diagnostics, and has been applied for virus detection. Though these methods have excellent sensitivity and specificity, most of them were not able to measure the quantity of virus. We here developed a droplet digital reverse transcription loop-mediated isothermal amplification ( R T-LAMP) e nhanced C as12b-based RNA d etection platform (RECD), for quantitative detection of viral RNA. CRISPR/Cas12b, which is more thermally stable than other family members in CRISPR systems, is combined with digital RT-LAMP. Due to the innate characteristic of digital format detection and CRISPR/Cas system, droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA, with single-molecule sensitivity. We expect the ddRECD assay will be a powerful tool for molecular diagnostics.

Background This study aimed to explore the possibility of serum tumor markers (TMs) combinations in assessing tumor metastasis in patients with lung cancer. Methods We performed a retrospective analysis of 541 patients diagnosed with lung cancer between January 2016 and December 2017 at the Pneumology Department of Dazhou Central Hospital. Serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA)125, CA153, CA199, CA724, cytokeratin 19 fragment (CYFRA), and neuron‐specific enolase (NSE) levels were quantified in each patient at the time of lung cancer diagnosis. Metastasis was confirmed by computed tomography, and/or positron emission tomography, and/or surgery or other necessary methods. Receiver operating characteristic (ROC) curves and calibration curves were used to evaluate the performance of the model. Results Of the 541 patients eligible for final analysis, 253 were detected with metastasis and 288 were detected without metastasis. Compared with those in nonmetastatic patients, the serum CEA, CA125, CA199, CA153, CYFRA, and NSE levels were notably higher in metastatic patients (P, The study developed a predictive model integrated with age, gender, and seven serum tumor markers (based on cut‐off value), which was efficient and reliable for assessing tumor metastasis before traditional standard methods are applied in highly suspected lung cancer patients. The predictive model combined with the decision tree model could provide some positive suggestions in the clinic.

Isolation and analysis of circulating tumor cells (CTCs) from the blood of patients at risk of metastatic cancers is a promising approach to improving cancer treatment. However, CTC isolation is difficult due to low CTC abundance and heterogeneity. Previously, we reported an ensemble-decision aliquot ranking (eDAR) platform for the rare cell and CTC isolation with high throughput, greater than 90% recovery, and high sensitivity, allowing detection of low surface antigen-expressing cells linked to metastasis. Here we demonstrate a sequential eDAR platform capable of isolating rare cells from whole blood with high purity. This improvement in purity is achieved by using a sequential sorting and flow stretching design in which whole blood is sorted and fluid elements are stretched using herringbone features and the parabolic flow profile being sorted a second time. This platform can be used to collect single CTCs in a multiwell plate for downstream analysis.

Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number. Here we test a digital format of NASBA (dNASBA) using a self-digitization (SD) chip platform, and apply it to quantifying HIV-1 RNA. We demonstrate that dNASBA is more sensitive and accurate than the real-time quantitative NASBA, and can be used to quantify HIV-1 RNA in plasma samples. Digital NASBA is thus a promising POC diagnostics tool for use in resource-limited settings.

Graphical abstract, The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called Cas12a-assisted RT-LAMP/AuNP (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/μL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.

Digital loop-mediated isothermal amplification (dLAMP) is attractive for the detection of nucleic acid due to its superior characteristics including isothermal amplification, absolute quantification, and single-molecule sensitivity. However, dLAMP suffers from the inaccurate quantification caused by low digital efficiency, which means only part of loaded template molecules could be amplified. We here developed a prehybridization-induced enhancement (PIE) strategy which could improve digital efficiency about 2-40 times without any new primer or additional operation. This work provides new insight into understanding the reaction dynamic of dLAMP. The PIE strategy could be applied to the other digital amplification methods.

As a key regulator, telomerase plays important roles in aging and tissue renewal and lifespan. It has been demonstrated that telomerase reactivation in adult or old mice can lead to tissue regeneration, delayed aging and significant lifespan extension. Therefore, modulation of telomerase activity in a precise manner has been received much attention. Here we develop a strategy using near infrared (NIR) modulation of telomerase activity based on gold nanocage@smart polymer system. By using this biocompatible design, cell migration, differentiation, cell senescence and cell cycle have been regulated. NIR-induced upregulation of telomerase can even protect cells from neurotoxic amyloid β induced cell death. This system has been used in vivo by taking advantages of NIR, such as good biocompatibility and tissues penetration. To the best of our knowledge, this is the first example for optical modulation of telomerase activity in living cells and tissues. In consideration of the important roles of telomerase in aging, cancer and hereditary disease, our work may inspire further application of multiple NIR-responsive control system for treatment of aging and related diseases.

Telomerase is a key regulator in cell metabolism, tissue renewal, and organismal lifespan. Here we develop a simple strategy to modulate cellular telomerase activity, and further control cell fate based on Mg2+ - and Zn2+ -activated DNAzymes in living cells. Through modulation of telomerase activity, we can regulate cell behavior, including cell migration, cell differentiation, cell senescence, and cell cycle. Our work provides a new way to modulate telomerase activity in living cells by using DNAzymes.

We developed an efficient cell surface engineering method based on lipid-insertion and host–guest recognition. With this methodology, we tailored cell membranes with β-cyclodextrin and subsequently manipulated cell behaviors through introducing photo-switchable guest molecule. Non-covalent nature afforded this method inherent reversibility. Furthermore, considering the remarkable molecular recognition property of aptamer, azobenzene-labeled aptamer was modified on cell surface through host–guest interaction and served as targeting ligand for selectively identifying target cells. Based on these, we designed a cell-based therapy for directing peripheral blood mononuclear cells to induce target cancer cell apoptosis. Our results provide new insights into engineering well-defined molecular recognition event on cell membrane to meet the demands of specific applications.

Meiosis, a cell division to generate gametes for sexual reproduction in eukaryotes, executes a single round of DNA replication and two successive rounds of chromosome segregation [1]. The extraordinary reliability of the meiotic cycle requires the activities of cyclin-dependent kinases (Cdks) associated with specific cyclins [2-4]. Cyclins are the regulatory subunits of protein kinases, which are the main regulators of maturation promoting factor or mitosis promoting factor (MPF) [5, 6] and anaphase-promoting complex/cyclosome (APC/C) [7, 8] in eukaryotic cell division. But how cyclins collaborate to control meiosis is still largely unknown. Cyclin B3 (Ccnb3) shares homology with A- and B-type cyclins [9], and is conserved during higher eukaryote evolution [10-17]. Previous studies have shown thatCcnb3-deleted females are sterile with oocytes unable to complete meiosis I inDrosophila[18], implying that Ccnb3 may have a special role in meiosis. To clarify the function of Ccnb3 in meiosis in mammalian species, we generatedCcnb3mutant mice by CRISPR/Cas9, and found thatCcnb3mutation caused female infertility with the failure of metaphase-anaphase transition in meiosis I. Ccnb3 was necessary for APC/C activation to initiate anaphase I, but not required for oocytes maturation, meiosis II progression, or early embryonic development. Our study reveals the differential cell cycle regulation between meiosis I and meiosis II, as well as meiosis between males and females, which shed light on the cell cycle control of meiosis.HighlightsIdentification of a female meiosis-specific cyclin in mouseCyclin B3 is required for metaphase-anaphase transition in oocyte meiosis ICyclin B3 is not essential for oocyte maturation and sister chromosome segregationCyclin B3 is necessary for APC/C activation and MPF kinase activity through Cdk1