Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

Brønsted Acid-Catalyzed Intramolecular Tandem Double Cyclization of γ-Hydroxy Acetylenic Ketones with Alkynes into Naphtho[1,2-b]furan-3-ones.

A metal-free and atom-economic route for the synthesis of naphtho[1,2-b]furan-3-ones has been realized via p-TsOH·H2O-catalyzed intramolecular tandem double cyclization of γ-hydroxy acetylenic ketones with alkynes in formic acid. The benzene-linked furanonyl-ynes are the key intermediates obtained by the scission/recombination of C-O double bonds. Further, the structural modifications of the representative product were implemented by reduction, demethylation, substitution, and [5 + 2]-cycloaddition.

Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance.

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.

Born approximation of trapping forces by acoustical Bessel and vortex fields.

Acoustic radiation forces have been used to trap various objects for fundamental studies and practical applications. Born approximation method, originally introduced to solve quantum scattering problems, is herein extended to analyze trapping forces exerted by two- and three-dimensional acoustic Bessel and vortex fields on spherical and nonspherical objects of arbitrary size. The results are compared with the conventional models like the partial wave expansion and Gorkov force potential. It is shown that for weakly scattering objects (such as common soft biological particles surrounded by fluids), the Born approximation can make predictions for the trapping forces on objects whose characteristic lengths are even up to multiple wavelengths of the sound beams. With the aid of the approximation, the Gorkov force potential is applied to analyze and gain insights into trapping forces on large objects far beyond the original Rayleigh scattering regime. The effects caused by the beam parameters, object shape, and orientation on the trapping behaviors are revealed. This work is useful for the further study of acoustic radiation forces and will guide the experiment of simplified acoustic tweezers on arbitrary-shaped particles.

Identification and validation of a fatty acid metabolism gene signature for the promotion of metastasis in liver cancer.

Metastasis is a fatal status for liver cancer, and the identification of an effective prediction model and promising therapeutic target is essential. Given the known relationship between fatty acid (FA) metabolism and the liver, the present study aimed to investigate dysregulation of genes associated with FA metabolism in liver cancer. Bioinformatics analyses were performed on data from patients with hepatocellular carcinoma (HCC) obtained from The Cancer Genome Atlas database using R software packages. Online public tools such as the Human Protein Atlas, Tumor Immune Single-Cell Hub and the University of Alabama at Birmingham Cancer Data Analysis portal were also utilized. Some essential results were further verified using in vitro experiments using HepG2 liver cancer cells. A signature consisting of three genes associated with the progression and prognosis of HCC and FA metabolism was identified. When samples were scored based on the expression of these genes and divided according to the median value, the higher score group showed a worse outcome and repressive immune microenvironment than the lower score group. Downstream pathways such as hypoxia, IL6/JAK/STAT3 and epithelial-mesenchymal transition were found to be significantly activated in the higher score group. As the core factor in the signature, mitochondrial ribosomal protein L35 (MRPL35) was found to be upregulated in HCC and to have certain impacts on the dysregulation of effective immunity. Further investigations and in vitro experiments indicated that MRPL35 facilitates the migration and invasion abilities of liver cancer, and the resistance of HCC to treatment. These findings have important implications regarding the characteristics and mechanisms of metastasis in liver cancer, and provide a promising signature based on FA metabolism-related genes that may be used to predict outcomes and explored as a novel therapeutic target in liver cancer.

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/µL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.

Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

Visualised genotyping assay with oral swabs in a closed tube by nested invasive reaction assisted with gold nanoparticle probes.

Single nucleotide polymorphism (SNP) typing is crucial for drug dosage and disease progression. Therefore, a simple and convenient genotyping assay is essential for personalised medicine. Herein, we developed a non-invasive, closed-tube, and visualised method for genotyping. In this method, oral swabs were lysed to directly perform PCR coupled with nested invasive reaction and visualisation based on gold nanoparticle probes in a closed tube. The strategy for genotyping assay depends on the single base recognition property of invasive reaction. This assay allowed quick and simple sample preparation and the detection of 25 copies/µL of CYP2C19*2 and 100 copies/µL of CYP2C19*3 within 90 min. Further, 20 oral swab samples for CYP2C19*2 and CYP2C19*3 were correctly typed, which agreed with pyrosequencing, indicating that this method has great potential for SNP typing in source-limited regions to guide personalised medicine.

A living biobank of matched pairs of patient-derived xenografts and organoids for cancer pharmacology.

Patient-derived tumor xenograft (PDX)/organoid (PDO), driven by cancer stem cells (CSC), are considered the most predictive models for translational oncology. Large PDX collections reflective of patient populations have been created and used extensively to test various investigational therapies, including population-trials as surrogate subjects in vivo. PDOs are recognized as in vitro surrogates for patients amenable for high-throughput screening (HTS). We have built a biobank of carcinoma PDX-derived organoids (PDXOs) by converting an existing PDX library and confirmed high degree of similarities between PDXOs and parental PDXs in genomics, histopathology and pharmacology, suggesting "biological equivalence or interchangeability" between the two. Here we demonstrate the applications of PDXO biobank for HTS "matrix" screening for both lead compounds and indications, immune cell co-cultures for immune-therapies and engineering enables in vitro/in vivo imaging. This large biobank of >550 matched pairs of PDXs/PDXOs across different cancers could become powerful tools for the future cancer drug discovery.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and ß within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Multiplexed and Rapid AST for Escherichia coli Infection by Simultaneously Pyrosequencing Multiple Barcodes Each Specific to an Antibiotic Exposed to a Sample.

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Tumor Purity in Preclinical Mouse Tumor Models.

Tumor biology is determined not only by immortal cancer cells but also by the tumor microenvironment consisting of noncancerous cells and extracellular matrix, together they dictate the pathogenesis and response to treatments. Tumor purity is the proportion of cancer cells in a tumor. It is a fundamental property of cancer and is associated with many clinical features and outcomes. Here we report the first systematic study of tumor purity in patient-derived xenograft (PDX) and syngeneic tumor models using next-generation sequencing data from >9,000 tumors. We found that tumor purity in PDX models is cancer specific and mimics patient tumors, with variation in stromal content and immune infiltration influenced by immune systems of host mice. After the initial engraftment, human stroma in a PDX tumor is quickly replaced by mouse stroma, and tumor purity then stays stable in subsequent transplantations and increases only slightly by passage. Similarly, in syngeneic mouse cancer cell line models, tumor purity also turns out to be an intrinsic property with model and cancer specificities. Computational and pathology analysis confirmed the impact on tumor purity by the diverse stromal and immune profiles. Our study deepens the understanding of mouse tumor models, which will enable their better and novel uses in developing cancer therapeutics, especially ones targeting tumor microenvironment. Significance: PDX models are an ideal experimental system to study tumor purity because of its distinct separation of human tumor cells and mouse stromal and immune cells. This study provides a comprehensive view of tumor purity in 27 cancers in PDX models. It also investigates tumor purity in 19 syngeneic models based on unambiguously identified somatic mutations. It will facilitate tumor microenvironment research and drug development in mouse tumor models.

Enhancing low-frequency water-column acoustic reflections in marine multichannel seismic data for seismic oceanography.

Reflections of low-frequency, broadband airgun signals from ocean water columns have long been collected in modern marine seismic surveys. However, they were barely examined because of their weak amplitude (following from low acoustic impedance contrast in water columns) and the lack of application interests. Nevertheless, in the past decades, a new cross-discipline "seismic oceanography" has developed the interest to use these water-column reflection signals to image the ocean structures. Motivated by seismic oceanography applications, we examined the acoustic multipath structure of marine seismic survey data with a focus on water-column reflections, and developed a two-step matched filtering approach to enhance water-column reflection and suppress the unwanted bubble waves. The approach was applied to process data collected from the Gulf of Mexico and led to an improvement in imaging mesoscale ocean structures when compared with the traditional matched filtering approach. For the specific data we examined, the results reveal a 11.3-dB improvement of signal-to-noise ratio by removing the noise and a 8-dB improvement of signal-to-reverberation ratio by suppressing bubble waves, while not affecting the information of ocean structures embedded in the signals. This study gains insights into features of water-column acoustic reflections and provides better tools for acoustic imaging of mesoscale ocean structures.

Phase shift approach for engineering desired radiation force: Acoustic pulling force example.

Phase shifts from scattering are used to analyze and engineer acoustic radiation forces. With the aid of phase shifts, analytical results for acoustic radiation forces can be simplified into compact and physically meaningful expressions, which can be used to develop a simplified procedure for the engineering of the radiation force. The desired radiation force can be fulfilled by a specific set of phase shifts up to a certain order, and then the required phase shifts can be fulfilled by engineering object and beam parameters. As an example, the phase shift approach is used to engineer the inner-to-outer radius ratio and the outer radius of a spherical shell to show how to use the phase shift method for the design of acoustic radiation forces. The example here is a force that is desired to pull particles against the propagation of a Bessel beam. A small paraxial parameter to pull a spherical shell is satisfied by in-phase scattering of monopole, dipole, quadrupole, octupole, and beyond. The example presented here is relatively simple yet reveals the advantages of the phase shift approach. The phase shift method can provide a simplified route for the design of acoustic tweezers using either traveling beams or standing waves.

MiR-590-5p inhibits pathological hypertrophy mediated heart failure by targeting RTN4.

Heart failure (HF) is a rising epidemic and public health burden in modern society. It is of great need to find new biomarkers to ensure a timely diagnosis and to improve treatment and prognosis of the disease. The mouse model of HF was established by thoracic aortic constriction. Color Doppler ultrasound was performed to detect left ventricular end-diastolic diameter. Hematoxylin and eosin staining was conducted to observe the pathological changes of mouse myocardium. The RT-qPCR analysis was performed to detect miR-590-5p and RTN4 expression levels. Western blot was conducted to detect protein levels of the indicated genes. We found that the expression of miR-590-5p was downregulated in cardiac tissues of HF mice. Injection of AAV-miR-590-5p attenuated myocardium hypertrophy and myocyte apoptosis. Additionally, miR-590-5p overexpression promoted viability, inhibited apoptosis, and decreased ANF, BNP and beta-MHC protein levels in H9c2 cell. Mechanistically, miR-590-5p binds to RTN4 3'-untranslated region, as predicted by starBase online database and evidenced by luciferase reporter assay. Furthermore, miR-590-5p negatively regulates RTN4 mRNA expression and suppresses its translation. The final rescue experiments revealed that miR-590-5p modulated cardiomyocyte phenotypes by binding to RTN4. In conclusion, miR-590-5p modulates myocardium hypertrophy and myocyte apoptosis in HF by downregulating RTN4.

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials ("Phase II-like mouse clinical trials"); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 µm cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 µM Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

Multiplex detection of blood-borne pathogens on a self-driven microfluidic chip using loop-mediated isothermal amplification.

Detection of blood-borne pathogens such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is essential to ensure the safety of blood transfusion. However, traditional PCR-based pathogen nucleic acid detection methods require relatively high experimental facilities and are difficult to apply in areas with limited resources. In this study, a self-driven microfluidic chip was designed to carry out multiplex detection of HBV, HCV and HIV by using loop-mediated isothermal amplification (LAMP). Benefitting from the air permeability of the polydimethylsiloxane material, the chip could accomplish sample loading within 12 min driven by the pressure difference between the reaction chambers and vacuum chambers in the chip without using pumps or any injection devices. Multiplex detection is achieved by presetting LAMP primers specific to different targets in different reaction chambers. Calcein was used as an indicator to indicate the positive amplification reaction, and the result can be recorded by a smartphone camera. After 50 min of isothermal amplification at 63 °C, 2 copies/µL of HBV, HCV and HIV target nucleic acids could be detected. The results of HBV detection of 20 clinical plasma samples by using the chip are consistent with that of the qPCR-based kit, indicating that the LAMP-based self-driven chip has the clinical application potential for blood-borne pathogen detection, especially in resource-limited areas.