ABSTRACT
A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.
Subject(s)
CRISPR-Cas Systems , MicroRNAs , MicroRNAs/analysis , MicroRNAs/blood , MicroRNAs/genetics , Humans , CRISPR-Cas Systems/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Limit of Detection , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Bacterial Proteins , Endodeoxyribonucleases , CRISPR-Associated ProteinsABSTRACT
Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.
Subject(s)
Anti-Bacterial Agents , CRISPR-Cas Systems , Gram-Negative Bacteria , Plasmids , CRISPR-Cas Systems/genetics , Plasmids/genetics , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/drug effects , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Gene Transfer Techniques , Gene Editing/methodsABSTRACT
Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.
Subject(s)
Cytokines , Mycobacterium tuberculosis , Cytokines/metabolism , Macrophages/microbiology , Macrophages/metabolism , Mycobacterium bovis , Mice , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Animals , Humans , NF-kappa B/metabolism , Microbial Viability , Bacterial AdhesionABSTRACT
Fast, sensitive, and portable detection of genetic modification contributes to agricultural security and food safety. Here, we developed RPA-CRISPR/Cas12a-G-quadruplex colorimetric assays that can combine with intelligent recognition by deep learning algorithms to achieve sensitive, rapid, and portable detection of the CaMV35S promoter. When the crRNA-Cas12a complex recognizes the RPA amplification product, Cas12 cleaves the G-quadruplex, causing the G4-Hemin complex to lose its peroxide mimetic enzyme function and be unable to catalyze the conversion of ABTS2- to ABTS, allowing CaMV35S concentration to be determined based on ABTS absorbance. By utilizing the RPA-CRISPR/Cas12a-G4 assay, we achieved a CaMV35S limit of detection down to 10 aM and a 0.01 % genetic modification sample in 45 min. Deep learning algorithms are designed for highly accurate classification of color results. Yolov5 objective finding and Resnet classification algorithms have been trained to identify trace (0.01 %) CaMV35S more accurately than naked eye colorimetry. We also coupled deep learning algorithms with a smartphone app to achieve portable and rapid photo identification. Overall, our findings enable low cost ($0.43), high accuracy, and intelligent detection of the CaMV35S promoter.
Subject(s)
CRISPR-Cas Systems , Colorimetry , Deep Learning , G-Quadruplexes , Colorimetry/methods , CRISPR-Cas Systems/genetics , Promoter Regions, Genetic , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , Limit of Detection , Bacterial Proteins/genetics , EndodeoxyribonucleasesABSTRACT
Mycophenolate mofetil (MMF) is commonly utilized for the treatment of neuromyelitis optica spectrum disorders (NMOSD). However, a subset of patients experience significant gastrointestinal (GI) adverse effects following MMF administration. The present study aims to elucidate the underlying mechanisms of MMF-induced GI toxicity in NMOSD. Utilizing a vancomycin-treated mouse model, we compiled a comprehensive data set to investigate the microbiome and metabolome in the GI tract to elucidate the mechanisms of MMF GI toxicity. Furthermore, we enrolled 17 female NMOSD patients receiving MMF, who were stratified into non-diarrhea NMOSD and diarrhea NMOSD (DNM) groups, in addition to 12 healthy controls. The gut microbiota of stool samples was analyzed using 16S rRNA gene sequencing. Vancomycin administration prevented weight loss and tissue injury caused by MMF, affecting colon metabolomes and microbiomes. Bacterial ß-glucuronidase from Bacteroidetes and Firmicutes was linked to intestinal tissue damage. The DNM group showed higher alpha diversity and increased levels of Firmicutes and Proteobacteria. The ß-glucuronidase produced by Firmicutes may be important in causing gastrointestinal side effects from MMF in NMOSD treatment, providing useful information for future research on MMF. IMPORTANCE: Neuromyelitis optica spectrum disorder (NMOSD) patients frequently endure severe consequences like paralysis and blindness. Mycophenolate mofetil (MMF) effectively addresses these issues, but its usage is hindered by gastrointestinal (GI) complications. Through uncovering the intricate interplay among MMF, gut microbiota, and metabolic pathways, this study identifies specific gut bacteria responsible for metabolizing MMF into a potentially harmful form, thus contributing to GI side effects. These findings not only deepen our comprehension of MMF toxicity but also propose potential strategies, such as inhibiting these bacteria, to mitigate these adverse effects. This insight holds broader implications for minimizing complications in NMOSD patients undergoing MMF therapy.
Subject(s)
Disease Models, Animal , Gastrointestinal Microbiome , Mycophenolic Acid , Neuromyelitis Optica , Mycophenolic Acid/adverse effects , Mycophenolic Acid/therapeutic use , Neuromyelitis Optica/drug therapy , Neuromyelitis Optica/microbiology , Humans , Animals , Mice , Gastrointestinal Microbiome/drug effects , Female , Adult , Middle Aged , Vancomycin/adverse effects , RNA, Ribosomal, 16S/genetics , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/microbiology , Diarrhea/chemically induced , Diarrhea/microbiology , Male , Gastrointestinal Diseases/chemically induced , Feces/microbiology , Bacteria/drug effects , Bacteria/genetics , Bacteria/classificationABSTRACT
Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20â¯mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20â¯mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.
Subject(s)
Colitis , Gallic Acid/analogs & derivatives , Gastrointestinal Microbiome , Mice , Animals , Mycophenolic Acid/pharmacology , Mycophenolic Acid/therapeutic use , Immunosuppressive Agents/therapeutic use , Glucuronidase/metabolism , Bacteria/metabolism , Colitis/drug therapyABSTRACT
Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , CRISPR-Cas Systems/genetics , Nucleic Acid Amplification Techniques/methods , Coloring Agents , ErbB Receptors/genetics , Biosensing Techniques/methodsABSTRACT
Simultaneous detection of multiple targets can provide important data support for clinical diagnosis and treatment. Here, we report a facile isothermal assay based on target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a (TM-RCT/Cas12a-Cas13a). Through facile one-step amplification (TM-RCT), two target DNAs are converted to RNA amplified products. The simultaneous detection of HPV16 and HPV18 is then achieved by combining two CRISPR/Cas systems. This system shows excellent sensing performance and provides a universal method for simultaneous detection.
ABSTRACT
Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , CRISPR-Cas Systems/genetics , Delayed-Action Preparations , RNA, Messenger/genetics , RNA , Coloring Agents , DNA, Single-Stranded/genetics , Endonucleases , Serine Proteinase InhibitorsABSTRACT
PURPOSE: The purpose of this study was to evaluate whether the new nickel-titanium alloy stents are superior to traditional silicone stents in hypospadias repair surgery to prevent complications such as urinary fistula. METHODS: This retrospective cohort study included 576 patients with hypospadias who underwent the placement either with nickel-titanium alloy stents or traditional silicone stents after hypospadias surgery between March 2002 and August 2019. The patients were assigned into the nickel-titanium alloy stent group (group NTAS) and the silicone stent group (group SS). The primary outcome was assessed with the rate of urinary fistula occurrence at four weeks (stent removal time), and the secondary outcomes were decided on the rate of other complications such as urethral stricture, and urethral diverticulum, infection, etc. The occurrence of complications in both groups was compared and the important contributing factors of urinary fistula and urethral stricture were determined. RESULTS: Among 576 patients, 398 were assigned into group NTAS while 178 were into group SS. 35 patients in the group NTAS and 30 in the group SS developed urinary fistula with a ratio of 8.8% and 16.9%, respectively (p = 0.005). Subgroup analysis showed that the differences were mainly in preschool patients (≤ 6 years) (p = 0.004) and those with the penile type of hypospadias (p = 0.008). In addition, urethral stricture complicated five patients in the NTAS group and two in the SS group with a ratio of 1.3% and 1.1%, respectively (p = 1.000). Logistic regression showed that hypospadias type (p = 0.001) and stent type (p = 0.001) are the important risk factors for urethral fistula. CONCLUSIONS: Nickel-titanium alloy stents reduced the occurrence of urinary fistula complications after hypospadias repair in preschool patients, and can be optioned as a better choice for hypospadias surgery.
ABSTRACT
MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.
Subject(s)
Biosensing Techniques , MicroRNAs , MicroRNAs/genetics , MicroRNAs/metabolism , CRISPR-Cas Systems , Nucleic Acid Amplification Techniques/methods , DNA, Single-Stranded , Biological Assay/methods , Biosensing Techniques/methodsABSTRACT
An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.
Subject(s)
CRISPR-Cas Systems , Genes, p53 , Humans , DNA Primers , Electrodes , FluorescenceABSTRACT
Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette-Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.
Subject(s)
MicroRNAs , Mycobacterium bovis , Tuberculosis , Humans , BCG Vaccine , THP-1 Cells , MicroRNAs/metabolism , Tuberculosis/microbiology , Mycobacterium bovis/metabolism , Apoptosis , Adenosine Triphosphatases , Chromosomal Proteins, Non-HistoneABSTRACT
DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3' end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10-4 U/mL and a broad detection range from 2 × 10-4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.
Subject(s)
Biosensing Techniques , Site-Specific DNA-Methyltransferase (Adenine-Specific) , CRISPR-Cas Systems , DNA Modification Methylases/metabolism , Coloring Agents , Biosensing Techniques/methods , DNA Methylation , DNA/genetics , DNA/metabolismABSTRACT
DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5'-phosphate terminuses. After treating with Lambda, the sequence with 5'-phosphate modification will be degraded, leaving the single-stranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.
Subject(s)
CRISPR-Associated Proteins , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , Endonucleases/genetics , Endonucleases/metabolism , CRISPR-Cas Systems/genetics , Recombinases/genetics , Recombinases/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , DNA Methylation , Bacterial Proteins/genetics , DNA, Single-Stranded/genetics , DNA/analysis , PhosphatesABSTRACT
The middle-aged male was diagnosed with "acute anterior wall myocardial infarction" based on clinical symptoms, laboratory examination, and coronary angiography (CAG), but his ECG showed no significant change in QRS wave or ST-T within 6 h of admission. Thus, a perfect explanation with the existing theory is difficult, and only the case is presented here.
Subject(s)
Anterior Wall Myocardial Infarction , Coronary Vessels , Male , Middle Aged , Humans , Coronary Vessels/diagnostic imaging , Electrocardiography , Coronary Angiography , HospitalizationABSTRACT
COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM-500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops.
Subject(s)
Biosensing Techniques , Nucleic Acid Amplification Techniques , Biosensing Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Limit of Detection , Nucleic Acid Amplification Techniques/methods , Promoter Regions, GeneticABSTRACT
Programmed cell death (PCD), including necroptosis, has emerged as a significant pathway in cardiovascular diseases. The infection of viral myocarditis (VMC) could cause cardiomyocytes degeneration, necrosis, and immune-inflammatory myocardial response. In this review, we summarized and evaluated the available evidence on the pathogenesis, molecule mechanism, diagnosis, and potential treatment strategies of viral myocarditis, with a special focus on the novel mechanism of necroptosis for cardiomyocytes death. Studies have shown that tumor necrosis factor-alpha (TNF-α) is an important cytokine involved in the activation of necroptosis; an elevated level of TNF-α is continually reported in patients suffering from VMC, implicating its involvement in the pathogenesis of VMC. It is of great interest to explore the clinical implication of TNF-α. We subsequently conducted a meta-analysis on the efficacy of serum TNF-α expression level and its diagnostic accuracy on acute viral myocarditis detection. Taken together, the review demonstrates a compelling role of necroptosis involved in the pathogenesis of VMC. Further, applying TNF-α as a serological marker for the diagnosis of VMC may be a useful strategy.
ABSTRACT
Docosahexaenoic acid (DHA) is a ω-3 polyunsaturated fatty acid, which can be uptaken by cells and is essential for proper neuronal and retinal function. However, the detailed physical impact of DHA molecules on the plasma membrane is still unclear. Hence, in this work, we carried out µs-scale coarse-grained molecular dynamics (MD) simulations to reveal the interactions between DHA molecules and a model cell membrane. As is known, the cell membrane can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains due to the differential interactions between lipids and proteins. In order to capture this feature, we adopted the three-component phase-separated lipid membranes and considered both anionic and neutral DHA molecules in the current work. Our results showed that DHA molecules can spontaneously self-assemble into nanoclusters, fuse with lipid membranes, and localize preferably in Ld membrane domains. During the membrane fusion process, DHA molecules can change the intrinsic transmembrane potential of the lipid membrane, and the effects of anionic DHA molecules are much more significant. Besides, the presence of DHA molecules mainly in the Ld membrane domains could regulate the differences in the lipid chain order, membrane thickness, cholesterol preference, and cholesterol flip-flop basically in a concentration-dependent manner, which further promote the stability of the intraleaflet dynamics and inhibit the interleaflet dynamics (or promote membrane domain registration) of the membrane domains. In short, the impact of DHA molecules on the physical properties of a model cell membrane on the molecular level revealed in our work will provide useful insights for understanding the biological functions of DHA molecules.