scholarly journals Autocatalytic–Protection for an Unknown Locus CRISPR–Cas Countermeasure for Undesired Mutagenic Chain Reactions

2020 ◽  
Author(s):  
Ethan Schonfeld ◽  
Elan Schonfeld ◽  
Dan Schonfeld
Keyword(s):  
Genomic Sequence ◽  
Homing Endonuclease ◽  
Gene Drive ◽  
Proof Of Concept ◽  
Chain Reaction ◽  
Gene Encoding ◽  
Chain Reactions ◽  
Homology Directed Repair ◽  
Novel Approach ◽  
Mutagenic Chain Reaction

AbstractThe mutagenic chain reaction (MCR) is a genetic tool to use a CRISPR–Cas construct to introduce a homing endonuclease, allowing gene drive to influence whole populations in a minimal number of generations1,2,3. The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele4? Thus far, MCR protection methods require knowledge of the terror locus5. Here we introduce a novel approach, an autocatalytic-Protection for an Unknown Locus (a-PUL), whose aim is to spread through a population and arrest and decrease an active terror event’s spread without any prior knowledge of the terror-modified locus, thus allowing later natural selection and ERACR drives to restore the normal locus6. a-PUL, using a mutagenic chain reaction, includes (i) a segment encoding a non-Cas9 endonuclease capable of homology-directed repair suggested as Type II endonuclease Cpf1 (Cas12a), (ii) a ubiquitously-expressed gene encoding a gRNA (gRNA1) with a U4AU4 3′-overhang specific to Cpf1 and with crRNA specific to some desired genomic sequence of non-coding DNA, (iii) a ubiquitously-expressed gene encoding two gRNAs (gRNA2/gRNA3) both with tracrRNA specific to Cas9 and crRNA specific to two distinct sites of the Cas9 locus, and (iv) homology arms flanking the Cpf1/gRNA1/gRNA2/gRNA3 cassette that are identical to the region surrounding the target cut directed by gRNA17. We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.

A Novel Approach of Plasma Nitrocarburizing Using a Solid Carbon Active Screen – a Proof of Concept

2017 ◽  
Vol 72 (5) ◽  
pp. 254-259 ◽  
Author(s):  
I. Burlacov ◽  
S. Hamann ◽  
H.-J. Spies ◽  
A. Dalke ◽  
J. Röpcke ◽  
...  
Keyword(s):  
Solid Carbon ◽  
Proof Of Concept ◽  
Novel Approach ◽  
Plasma Nitrocarburizing ◽  
Active Screen

scholarly journals Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

2021 ◽  
Vol 9 (7) ◽  
pp. 1463
Author(s):  
Tamirat Tefera Temesgen ◽  
Kristoffer Relling Tysnes ◽  
Lucy Jane Robertson
Keyword(s):  
Oxidative Stress ◽  
Stress Responses ◽  
Target Genes ◽  
Cryptosporidium Oocyst ◽  
Drinking Water Treatment ◽  
Proof Of Concept ◽  
Environmental Matrices ◽  
Oocyst Wall ◽  
Novel Approach ◽  
Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

scholarly journals Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
François Stüder ◽  
Jean-Louis Petit ◽  
Stefan Engelen ◽  
Marco Antonio Mendoza-Parra
Keyword(s):  
Real Time ◽  
Optimal Solution ◽  
Proof Of Concept ◽  
Gene Encoding ◽  
Spike Gene ◽  
Long Period ◽  
The World ◽  
Receptor Recognition ◽  
Nanopore Dna Sequencing ◽  
Novel Coronavirus

AbstractSince December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants’ occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants’ tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants’ emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant’s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant’s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

scholarly journals IκBα targeting promotes oxidative stress-dependent cell death

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Giovanna Carrà ◽  
Giuseppe Ermondi ◽  
Chiara Riganti ◽  
Luisella Righi ◽  
Giulia Caron ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Protein Interactions ◽  
In Silico ◽  
Inverse Correlation ◽  
Ros Production ◽  
Gene Encoding ◽  
Specific Expression ◽  
Novel Approach ◽  
Stress Dependent

Abstract Background Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy. Methods We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions. Results Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis. Conclusions NFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.

scholarly journals Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122 ◽  
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

scholarly journals Modeling the Manipulation of Natural Populations by the Mutagenic Chain Reaction

Genetics ◽  
2015 ◽  
Vol 201 (2) ◽  
pp. 425-431 ◽  
Author(s):  
Robert L. Unckless ◽  
Philipp W. Messer ◽  
Tim Connallon ◽  
Andrew G. Clark
Keyword(s):  
Natural Populations ◽  
Chain Reaction ◽  
Mutagenic Chain Reaction

Investigation of Mutations in Exon 12 Of MYH7, 16 Of β-MYBPC3 and 2 Of TCAP Gene in Dogs with Dilated Cardiomyopathy using PCR-SSCP Technique

2021 ◽  
Author(s):  
R.B. Vishnurahav ◽  
S. Ajithkumar ◽  
Usha Narayana Pillai ◽  
N. Madhvan Unny ◽  
K.D. John Martin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dilated Cardiomyopathy ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Multifunctional Protein ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Intron 1 ◽  
Cardiac Disorders ◽  
Polymerase Chain

Background: Dilated cardiomyopathy is the important myocardial disease and one of the most common cause of death in the medium to large size dog breeds worldwide. The disease is characterized by dilatation of cardiac chambers and thinning of walls leads to systolic failure. Mutations in some sarcomere genes leads to cardiomyopathy in humans. Sarcomere is an important multifunctional protein network involved in the signal reception and transduction. Mutations in β-MYH7, MYBPC3 and TCAP genes produce alterations in the morphology of heart (hypertrophy or dilatation).Methods: In this study twenty apparently healthy and twenty five dogs with dilated cardiomyopathy (DCM) were selected from patients reported or referred to University Veterinary Hospital and Teaching Veterinary Clinical Complex, Mannuthy (2015-2017) based on the clinical examination, radiographic, electrocardiographic, haematobiochemical and echocardiographic studies cardiac disorders (Dilated cardiomyopathy and hypertrophic cardiomyopathy) were confirmed.Result: In the present study we investigated genetic alterations of exon 12 of MYH7, 16 of β-MYBPC3 and 2 of TCAP gene in dogs by polymerase chain reaction -single stranded confirmation of polymorphism (PCR-SSCP). Polymerase chain reactions were analysed using acrylamide gel and samples with different pattern of bands were sequenced. Polymerase chain reaction-SSCP showed different migration of band pattern in the intron 1 of TCAP gene in one sample.

Practicality Assessment of a Frugal Nanoparticle Generator: Opening doors to Nano Pharmaceuticals for the researchers and students of underprivileged academic institutions.

2021 ◽  
Vol 17 ◽  
Author(s):  
Swayamprakash Patel ◽  
Ashish Patel ◽  
Mruduka Patel ◽  
Umang Shah ◽  
Mehul Patel ◽  
...  
Keyword(s):  
Zeta Potential ◽  
Organic Solvent ◽  
Ultrasonic Vibration ◽  
High Speed ◽  
Patent Application ◽  
Entrapment Efficiency ◽  
Proof Of Concept ◽  
Research Institutions ◽  
Novel Approach ◽  
Sophisticated Equipment

Background: Probe sonication and High-speed homogenizer are comparatively costly equipment to fabricate the nanoparticles. Many academic and research institutions cannot afford the procurement and maintenance of such sophisticated equipment. In the present work, a newer idea is conceptualized, which can be adopted by the underprivileged research institutions to fabricate solid lipid nanoparticles (SLN) in the absence of sophisticated equipment. The current work describes the pilot-level trials of this novel approach. This study represents the preliminary proof-of-concept trials for which the Indian patent application (3508/MUM/2015) is filed. Method: A frugal piece of equipment was made using a 50 ml centrifuge tube with conical bottom and a piezoelectric mist maker or humidifier. SLNs were prepared by combining the quasi-emulsion solvent evaporation approach and ultrasonic vibration approach. A quasi-emulsion was composed by the dropwise mixing of the organic solvent containing drug & lipid with an aqueous solution containing surfactant under continuous ultrasonic vibration in the piezoelectric chamber. The size of the droplets was significantly reduced due to piezoelectric ultrasonic vibration. Under the provision of mild vacuum and heat generated by vibration, the organic solvent was evaporated, which leaves behind a suspension of SLN. In the present work, albendazole was selected as a model drug. Various trials with Compritol 888 ATO® and Precirol ATO 5® as a lipid carrier and Tween 80 and Poloxamer 188 as a surfactant were performed. Zeta potential of SLNs was improved by the addition of polyelectrolytes like K2SO4 and Na4P2O7. Result and Conclusion: The ratio of drug to lipid was optimized to 1:4 for the most favorable results. SLN with a minimum Z-average diameter of 98.59 nm, -21 mV zeta potential, and 34.064 % (SD 10.78, n=9) entrapment efficiency were developed using the Precirol ATO 5 ® as a lipid carrier. The proof of concept for this novel approach is established through the development of Albendazole SLNs. This approach must also be evaluated for the development of polymeric nanoparticles and vesicular formulations. The further sophistication of the frugal equipment may allow more control over the quality of SLN. This approach will enable underprivileged researchers to prepare Nanopharmaceuticals. Researchers and students of such institutions can focus on the application of SLN by resolving the constraint of sophisticated equipment with this novel approach. This novel approach should also be tried for polymeric and vesicular nanopharmaceuticals.

scholarly journals Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus

2019 ◽  
Vol 70 (3) ◽  
pp. 464-473 ◽  
Author(s):  
Won-Keun Kim ◽  
Jin Sun No ◽  
Daesang Lee ◽  
Jaehun Jung ◽  
Hayne Park ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Disease Risk ◽  
Hemorrhagic Fever ◽  
Epidemiological Surveillance ◽  
Likelihood Method ◽  
Comparative Genomic ◽  
Novel Approach ◽  
Epidemiological Surveys ◽  
Outbreak Areas ◽  
Complete Sequences

Abstract Background Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. Methods Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction–based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. Results The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. Conclusions This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

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