scholarly journals A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics

2021 ◽  
Vol 7 (5) ◽  
pp. eabc7802
Author(s):  
Kai Shi ◽  
Shiyi Xie ◽  
Renyun Tian ◽  
Shuo Wang ◽  
Qin Lu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genomic Dna ◽  
Reaction Network ◽  
Clinical Samples ◽  
Great Promise ◽  
Base Specificity ◽  
Nucleotide Mutation ◽  
Positive Feedback Circuit ◽  
Stringent Target ◽  
And Function

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

scholarly journals Self-resetting Molecular Probes for Nucleic Acids Enabled by Fuel Dissipative Systems

2021 ◽  
Author(s):  
Na Li ◽  
Yu Liu ◽  
Zhe Yin ◽  
Rui Liu ◽  
Linghao Zhang ◽  
...  
Keyword(s):  
Public Health ◽  
Nucleic Acid ◽  
Disease Control ◽  
Dna Nanotechnology ◽  
Dissipative System ◽  
Molecular Probes ◽  
Dissipative Systems ◽  
Clinical Samples ◽  
Global Public Health ◽  
Nucleotide Mutation

Amid of COVID-19 pandemic devastating the public health around the world, it become urgent to maintain a sufficiently large supply of nucleic acid tests to screen suspected cases timely. Reusable molecular probes in current testing method could potentially lead to enormous amount of screening capacity, critical for the disease control. Herein, we for the first time report a kind of self-resetting molecular probes for repeatedly detecting SARS-CoV-2 RNA, enabled by orchestrating a biomimetic fuel dissipative system via dynamic DNA nanotechnology. A set of simulation toolkits was utilized for the design and optimization of the self-resetting probe, allowing for highly consistent signal amplitudes across cyclic detections of SARS-CoV-2 RNA. Idiosyncratically, FWHM regulated by dissipative kinetics exhibits a fingerprint signal for high confidential identification of single-nucleotide mutation in the virus sequence. We further exploited our self-resetting probes to examine multiple human-infectious RNA virus including SARS-CoV-2, ZIKV, MERS-CoV, and SARS-CoV to demonstrate its generic nucleic acid detection capability and superior orthogonality. Self-resetting probes were also deployed for detection of 110 clinical nasopharyngeal swabs and correctly classify all the clinical samples from 55 COVID-19 patients and 55 controls. We anticipate that the DNA nanotechnology-enabled self-resetting probe could circumvent the lack of sustainability in the diagnostics of COVID-19 and other infectious diseases, thus helping disease control and building a broader global public health agenda.

scholarly journals Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins

2021 ◽  
Vol 22 (5) ◽  
pp. 2647
Author(s):  
M. Quadir Siddiqui ◽  
Maulik D. Badmalia ◽  
Trushar R. Patel
Keyword(s):  
Nucleic Acid ◽  
Nuclear Export ◽  
Consensus Sequence ◽  
Nuclear Export Signal ◽  
Bioinformatic Analysis ◽  
Nucleic Acid Binding ◽  
Protein Protein Interaction ◽  
Lim Domains ◽  
Protein Nucleic Acid ◽  
And Function

Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.

scholarly journals Rapid Molecular Diagnosis of Tuberculosis and Its Resistance to Rifampicin and Isoniazid with Automated MDR/MTB ELITe MGB® Assay

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 797
Author(s):  
Vichita Ok ◽  
Alexandra Aubry ◽  
Florence Morel ◽  
Isabelle Bonnet ◽  
Jérôme Robert ◽  
...  
Keyword(s):  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Clinical Samples ◽  
Great Promise ◽  
Routine Practice ◽  
Pcr Assay ◽  
Perfect Agreement ◽  
Mdr Tb ◽  
Good Agreement

The MDR/MTB ELITe MGB® kit (ELITech) carried on the ELITe InGenius® platform is a new real-time PCR assay allowing automated extraction and detection of DNA of the Mycobacterium tuberculosis complex (MTB) and mutations in the rpoB and katG genes and inhA promoter region (pro-inhA) associated to resistance to rifampicin and isoniazid, the two markers of multidrug-resistant TB (MDR). We assessed the performances of the test on a collection of strains (n = 54) and a set of clinical samples (n = 242) from routine practice, comparatively to TB diagnosis and genotypic drug susceptibility testing (gDST) as references. Regarding the 242 clinical samples, the sensitivity and specificity of MTB detection by ELITe were 90.9% and 97.5%, respectively. For the detection of resistance-conferring mutations on positive clinical samples, we observed perfect agreement with gDST for katG and pro-inhA (κ = 1.0) and two discordant results for rpoB (κ = 0.82). Considering the 54 cultured strains, very good agreement with gDST was observed for the detection of the 25 distinct mutations in rpoB, katG, and pro-inhA, (κ = 0.95, 0.88, and 0.95, respectively). In conclusion, the automated MDR/MTB ELITe MGB® assay shows great promise and appears to be a valuable tool for rapid detection of pre-MDR- and MDR-TB directly from clinical specimens.

DNA Nanosieve-Based Regenerative Electrochemical Biosensor Utilizing Nucleic Acid Flexibility for Accurate Allele Typing in Clinical Samples

ACS Sensors ◽  
2021 ◽  
Vol 6 (3) ◽  
pp. 1348-1356
Author(s):  
Jin-Yuan Chen ◽  
Liang-Yong Yang ◽  
Zhou-Jie Liu ◽  
Qing-Xia Wei ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Electrochemical Biosensor ◽  
Clinical Samples

scholarly journals CRISPR is a useful biological tool for detecting nucleic acid of SARS-CoV-2 in human clinical samples

2021 ◽  
pp. 111772
Author(s):  
Md. Rashidur Rahman ◽  
Md. Amjad Hossain ◽  
Md. Mozibullah ◽  
Fateh Al Mujib ◽  
Afrina Afrose ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clinical Samples ◽  
Biological Tool

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

ChemInform Abstract: Applications of Psoralens as Probes of Nucleic Acid Structure and Function

ChemInform ◽  
2010 ◽  
Vol 22 (16) ◽  
pp. no-no
Author(s):  
Y. B. SHI ◽  
S. E. LIPSON ◽  
D. Y. CHI ◽  
H. P. SPIELMANN ◽  
J. A. MONFORTE ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Structure And Function ◽  
Nucleic Acid Structure ◽  
And Function ◽  
Acid Structure

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Cytological and Biochemical Studies of Anucleolate Xenopus Larvae

1962 ◽  
Vol s3-103 (61) ◽  
pp. 25-35
Author(s):  
H. WALLACE
Keyword(s):  
Alkaline Phosphatase ◽  
Nucleic Acid ◽  
Acid Content ◽  
Deoxyribonucleic Acid ◽  
Nucleic Acid Content ◽  
Tail Bud ◽  
Lethal Mutant ◽  
Cytoplasmic Rna ◽  
Biochemical Studies ◽  
And Function

A larval lethal mutant of Xenopus laevis lacks true nucleoli but possesses analogous intranuclear organelles, here termed blobs, which are smaller and more numerous than nucleoli. Cytochemical tests reveal that blobs (like nucleoli) contain ribonucleic acid (RNA), arginine, and alkaline phosphatase, but probably no Feulgen-positive material. Anucleolate larvae are deficient in cytoplasmic RNA. By biochemical methods the nucleic acid content of anucleolate embryos is found to be normal at the tail-bud stage, but does not increase after this. By the time they hatch, anucleolate larvae are deficient in both RNA and deoxyribonucleic acid (DNA). The implications of this and related mutations on the formation and function of the nucleolus are considered. The term ‘blob’ is justified in that it would be misleading to regard such organelles as nucleoli produced by normally latent organizers.

scholarly journals Innovative Therapeutic Approaches for Huntington’s Disease: From Nucleic Acids to GPCR-Targeting Small Molecules

2021 ◽  
Vol 15 ◽  
Author(s):  
Hidetoshi Komatsu
Keyword(s):  
Nucleic Acid ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
High Throughput Screening ◽  
Neurodegenerative Disorder ◽  
Direct Injection ◽  
Cag Repeat ◽  
Great Promise ◽  
Nucleic Acid Delivery ◽  
Small Interfering Rnas

Huntington’s disease (HD) is a fatal neurodegenerative disorder due to an extraordinarily expanded CAG repeat in the huntingtin gene that confers a gain-of-toxic function in the mutant protein. There is currently no effective cure that attenuates progression and severity of the disease. Since HD is an inherited monogenic disorder, lowering the mutant huntingtin (mHTT) represents a promising therapeutic strategy. Huntingtin lowering strategies mostly focus on nucleic acid approaches, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). While these approaches seem to be effective, the drug delivery to the brain poses a great challenge and requires direct injection into the central nervous system (CNS) that results in substantial burden for patients. This review discusses the topics on Huntingtin lowering strategies with clinical trials in patients already underway and introduce an innovative approach that has the potential to deter the disease progression through the inhibition of GPR52, a striatal-enriched class A orphan G protein-coupled receptor (GPCR) that represents a promising therapeutic target for psychiatric disorders. Chemically simple, potent, and selective GPR52 antagonists have been discovered through high-throughput screening and subsequent structure-activity relationship studies. These small molecule antagonists not only diminish both soluble and aggregated mHTT in the striatum, but also ameliorate HD-like defects in HD mice. This therapeutic approach offers great promise as a novel strategy for HD therapy, while nucleic acid delivery still faces considerable challenges.

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