Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic ...Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs.
Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs).
Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10–30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals.
This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.
This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
•Unnatural base pairs used in semi-synthetic organism were electrochemically analyzed.•Unnatural thioamide nucleobases provided catalytic hydrogen evolution reaction.•Unnatural base specific ...catalytic signals detected using mercury or amalgam electrode.•Method sensitive enough to identify single unnatural base in intact plasmid DNA.•Electroanalysis is suitable for advancement of synthetic biology.
While standard Watson-Crick base pairing normally involves hydrogen bonds that join size complementary nitrogenous heterocycles, certain unnatural base pairs (UBP) can support replication, transcription, and translation, including these in semi-synthetic organisms (SSO), using size complementarity alone. In this paper we show that the UBPs can be analyzed electrochemically as components of ribo- or 2′-deoxyribonucleosides, corresponding (2’-deoxy)triphosphates, as well as incorporated into DNA. Here, performance of the electrochemical methods profits from the ability of the unnatural bases 5SICS and TPT3 adsorbed at the surface of a mercury electrode to catalyze hydrogen evolution in acidic media. This allows semi-quantitative detection of single UBP within plasmid DNA generated in a SSO. This electrochemical approach provides new way for direct, fast, and low cost UBP analysis and can supplement or replace currently used methods of UBP detection.
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Mitochondrially targeted anticancer drugs (mitocans) that disrupt the energy-producing systems of cancer are emerging as new potential therapeutics. Mitochondrially targeted tamoxifen (MitoTam), an ...inhibitor of mitochondrial respiration respiratory complex I, is a first-in-class mitocan that was tested in the phase I/Ib MitoTam-01 trial of patients with metastatic cancer. MitoTam exhibited a manageable safety profile and efficacy; among 37% (14/38) of responders, the efficacy was greatest in patients with metastatic renal cell carcinoma (RCC) with a clinical benefit rate of 83% (5/6) of patients. This can be explained by the preferential accumulation of MitoTam in the kidney tissue in preclinical studies. Here we report the mechanism of action and safety profile of MitoTam in a case series of RCC patients. All six patients were males with a median age of 69 years, who had previously received at least three lines of palliative systemic therapy and suffered progressive disease before starting MitoTam. We recorded stable disease in four, partial response in one, and progressive disease (PD) in one patient. The histological subtype matched clear cell RCC (ccRCC) in the five responders and claro-cellular carcinoma with sarcomatoid features in the non-responder. The number of circulating tumor cells (CTCs) was evaluated longitudinally to monitor disease dynamics. Beside the decreased number of CTCs after MitoTam administration, we observed a significant decrease of the mitochondrial network mass in enriched CTCs. Two patients had long-term clinical responses to MitoTam, of 50 and 36 weeks. Both patients discontinued treatment due to adverse events, not PD. Two patients who completed the trial in November 2019 and May 2020 are still alive without subsequent anticancer therapy. The toxicity of MitoTam increased with the dosage but was manageable. The efficacy of MitoTam in pretreated ccRCC patients is linked to the novel mechanism of action of this first-in-class mitochondrially targeted drug.
•A simple label-free technique for analysis of nucleic acid polymerization mixtures.•The technique is based on selective removal of interfering monomers from electrode.•Monomers are removed by ...treatment with a surfactant or water at elevated temperature.
Previously it has been shown that cyclic nucleoside monophosphates can spontaneously polymerize to form RNA oligonucleotides under conditions simulating prebiotic conditions on Archean Earth. The most efficient polymerization was documented with 3′,5′-cyclic guanosine monophosphate (cGMP). In this work a method for fast detection of short polyG RNAs present in a large overabundance of cGMP, modeling conditions in the non-enzymatic nucleotide polymerization mixtures, is presented. The method is based on electrochemical measurements of guanine (G) oxidation signals yielded by RNA oligomers adsorbed onto the surface of a pyrolytic graphite electrode (PGE). To avoid false positive results arising from the G oxidation signals due to co-adsorbed cGMP, a method for selective removal of the monomers from the electrode surface has been devised. In the first step, both cGMP and RNAs are co-adsorbed onto the PGE surface. In the second step, the cGMP is selectively desorbed using treatments in solutions of different tested surfactants (SDS, Tween 20 or Triton X-100), or by washing in deionized water at elevated temperature. We show that this new approach is suitable for selective analysis of products of polymerization reactions from mixtures of their building blocks.
Recently we have demonstrated that pyrolytic graphite electrodes (PGE) offer a broad analytically useful potential window, ranging from about −2.0 V to +1.6 V vs. Ag|AgCl|3 M KCl, which enables to ...use the PGE not only for anodic measurements, but also for direct reduction of nucleobases in DNA oligonucleotides. In this follow‐up study, we have focused on the electrochemical behavior of four 2′‐deoxynucleosides derived from adenine, guanine, cytosine, thymine, uracil, and 5‐methyl cytosine on the PGE. On one hand we have obtained analogous primary redox responses as previously with the oligonucleotides. On the other hand, significant differences were observed, particularly when considering secondary responses involving products of the primary conversions, suggesting involvement of different mechanisms. Further we have found that presence of the ambient oxygen in the electrolyte does not dramatically affect the redox signals of the nucleosides. This finding is in contrast with DNA responses measured at the mercury‐based electrodes, where deaeration prior to the measurements was necessary. We demonstrate that all studied nucleosides can be analyzed using a simple ex situ (medium exchange) procedure.
Before the first humans depart for Mars in the next decade, hundreds of tons of martian water-ice must be harvested to produce propellant for the return vehicle, a process known as
resource ...utilization (ISRU). We describe here an instrument, the Agnostic Life Finder (ALF), that is an inexpensive life-detection add-on to ISRU. ALF exploits a well-supported view that informational genetic biopolymers in life in water must have two structural features: (1) Informational biopolymers must carry a repeating charge; they must be
(2) Their building blocks must fit into an
the building blocks must be size-shape regular. ALF exploits the first structural feature to extract polyelectrolytes from ∼10 cubic meters of mined martian water by applying a voltage gradient perpendicularly to the water's flow. This gradient diverts polyelectrolytes from the flow toward their respective electrodes (polyanions to the anode, polycations to the cathode), where they are captured in cartridges before they encounter the electrodes. There, they can later be released to analyze their building blocks, for example, by mass spectrometry or nanopore. Upstream, martian cells holding martian informational polyelectrolytes are disrupted by ultrasound. To manage the (unknown) conductivity of the water due to the presence of salts, the mined water is preconditioned by electrodialysis using porous membranes. ALF uses only resources and technology that must
be available for ISRU. Thus, life detection is easily and inexpensively integrated into SpaceX or NASA ISRU missions.
An innovative approach to label-free voltammetric analysis of DNA at a pyrolytic graphite electrode (PGE) within a broad range of potentials (from −2.0 to +1.6V) in an acetate buffer (pH5) is ...presented. Using specifically designed DNA nonamers, we demonstrate not only anodic oxidation, but for the first time also cathodic reduction of nucleobases at the PGE. In addition, products of irreversible oxidation/reduction of the parent bases are shown to yield analytically useful, base-specific cathodic/anodic signals, making it possible to distinguish between the canonical bases (adenine, cytosine, guanine and thymine), uracil (U) and 5-methylcytosine (mC) in DNA. Furthermore, selective electrochemical “switching off” of the redox signals specific to certain nucleobases is presented as a way to resolve overlapping signals. Similarly, newly reported signals corresponding to electrochemically transformed bases can be “switched on” under specific conditions. This approach can be utilized for fast and facile simultaneous label-free analysis of bases in DNA, including mC and U, and to uncover overlapping signals. This significantly extends the possible applications of PGE in DNA research and (bio)sensor development.
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•First use of a pyrolytic graphite electrode (PGE) for electroreduction of DNA bases•PGE offers DNA redox signals analogous to those of mercury-based electrodes.•Novel analytically applicable redox signals of DNA bases were found.•Distinct cathodic signals of A, C, G, T, mC or U can be measured at the PGE.•Cathodic polarization can selectively switch off DNA anodic signals and vice versa.
•Electroanalysis of Artificially Expanded Genetic Information System (AEGIS).•Electroactivity of 8 AEGIS 2’-deoxynucleosides was described of which 1 was inactive.•Pyrolytic graphite electrode was ...used for reductions at highly negative potentials.•Pyrolytic graphite electrode was used for oxidations at highly positive potentials.•Newly described peaks will be later used for AEGIS base identification in DNA.
Recently we showed the reduction and oxidation of six natural 2′-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2′-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.
In this paper we present a rapid electrochemical enzyme-linked DNA hybridization assay using disposable pencil graphite electrodes (PeGE) to detect target DNA (tDNA) sequences in DNA fragments ...amplified by polymerase chain reaction. The procedure consists of several short (1–2 min) incubation steps, including adsorption of the tDNA at unpretreated PeGE from denaturing medium, surface blocking with milk proteins, hybridization with a biotinylated oligonucleotide probe and binding of streptavidin-alkaline phosphatase conjugate to the biotin tags. Then the PeGE is transferred into background electrolyte solution containing 1-naphthyl phosphate, which is enzymatically dephosphorylated to give electrochemically oxidizable indicator 1-naphthol. The assay, which can be performed within 7–8 min, offers a perfect discrimination between specific and nonspecific DNA amplicons and easy detection of about ~40 femtomoles of tDNA in large excesses of non-complementary DNA. An application on the detection of p53 gene deletion in a human cell culture, featuring a real biological model, is presented. The designed setup has a potential to be applied as one of simple, fast, robust ultralow cost “do-it-yourself “instruments recently introduced as diagnostic tools for third world countries.
The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The ...aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I–III colon cancer.
An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I–III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance.
Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0·97 for colon tumour; r=0·97 for invasive margin; p<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 8% patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio HR for high vs low Immunoscore 0·20, 95% CI 0·10–0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21–0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system.
The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer.
French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.
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