• Some of these testing processes will enable the administrator to: – Record and retransmit packets from real virus or worm scan – Record and retransmit packets from a real virus or worm scan with incomplete TCP/IP session connections (missing SYN packets) – Conduct a real virus or worm scan against an invulnerable system Principles of ...
houston journal of international law touched by an agent: why the united states should look to the rest of the world for a new airport security scheme and stop using full-body scanners. Retrieved from &sid=3a528928-b040-4f46-bf06-4e110473738e%40sessionmgr102 (N.A.), (N.D.).
Aug 07, 2020 · CRISPR/Cas9 is a simple two-component system used for effective targeted gene editing. The first component is the single-effector Cas9 protein, which contains the endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complementary to the spacer sequence and HNH cleaves the complementary strand.
Aug 13, 2020 · Extensive data on cultivable virus from asymptomatic or presymptomatic individuals are lacking, with one outbreak investigation in a care home reporting detection of cultivable virus in one asymptomatic and 17 presymptomatic cases . Although we saw a higher proportion of asymptomatic cases in the age group 81–100 years, the reasons and ...
A major concern for implementing CRISPR/Cas9 for gene therapy is the relatively high frequency of off-target effects (OTEs), which have been observed at a frequency of ≥50% ( 31 ). Current attempts at addressing this concern include engineered Cas9 variants that exhibit reduced OTE and optimizing guide designs. One strategy that minimizes OTEs utilizes Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs), in combination with an sgRNA pair targeting both strands of the DNA at the intended location to produce the DSB ( 32 ). Researchers have also developed Cas9 variants that are specifically engineered to reduce OTEs while maintaining editing efficacy ( Table 1 ). SpCas9-HF1 is one of these high-fidelity variants that exploits the “excess-energy” model which proposes that there is an excess affinity between Cas9 and target DNA which may be enabling OTEs. By introducing mutations to 4 residues involved in direct hydrogen bonding between Cas9 and the phosphate backbone of the target DNA, SpCas9-HF1 has been shown to possess no detectable off-target activity in comparison to wildtype SpCas9 ( 35 ). Other Cas9 variants that have been developed include evoCas9 and HiFiCas9, both of which contain altered amino acid residues in the Rec3 domain which is involved in nucleotide recognition. Desensitizing the Rec3 domain increases the dependence on specificity for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy ( 38, 39 ). One of the more recent developments is the Cas9_R63A/Q768A variant, in which the R63A mutation destabilizes R-loop formation in the presence of mismatches and Q768A mutation increases sensitivity to PAM-distal mismatches ( 49 ). Despite the different strategies, the rational for generating many Cas9 variants with reduced OTEs has been to ultimately reduce general Cas9 and DNA interactions and give a stronger role for the DNA:RNA heteroduplex in facilitating the edits.
The birth of gene therapy as a therapeutic avenue began with the repurposing of viruses for transgene delivery to patients with genetic diseases. Gene therapy enjoyed an initial phase of excitement, until the recognition of immediate and delayed adverse effects resulted in death and caused a major setback. More recently, the discovery and development of CRISPR/Cas9 has re-opened a door for gene therapy and changed the way scientists can approach a genetic aberration—by fixing a non-functional gene rather than replacing it entirely, or by disrupting an aberrant pathogenic gene. CRISPR/Cas9 provides extensive opportunities for programmable gene editing and can become a powerful asset for modern medicine. However, lessons learned from traditional gene therapy should prompt greater caution in moving forward with CRISPR systems to avoid adverse events and setbacks to the development of what may be a unique clinically beneficial technology. A failure to take these lessons into account may provoke further backlash against CRISPR/Cas9 development and slow down progression toward attaining potentially curative gene editing technologies.
The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.
CR has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Astra Zeneca , Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo, and Pharmar, and is on the scientific advisory boards of Harpoon Therapeutics and Bridge Medicines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The bacterial CRISPR locus was first described by Francisco Mojica ( 23) and later identified as a key element in the adaptive immune system in prokaryotes ( 24 ). The locus consists of snippets of viral or plasmid DNA that previously infected the microbe (later termed “spacers”), which were found between an array of short palindromic repeat sequences. Later, Alexander Bolotin discovered the Cas9 protein in Streptococcus thermophilus, which unlike other known Cas genes, Cas9 was a large gene that encoded for a single-effector protein with nuclease activity ( 25 ). They further noted a common sequence in the target DNA adjacent to the spacer, later known as the protospacer adjacent motif (PAM)—the sequence needed for Cas9 to recognize and bind its target DNA ( 25 ). Later studies reported that spacers were transcribed to CRISPR RNAs (crRNAs) that guide the Cas proteins to the target site of DNA ( 26 ). Following studies discovered the trans-activating CRISPR RNA (tracrRNA), which forms a duplex with crRNA that together guide Cas9 to its target DNA ( 27 ). The potential use of this system was simplified by introducing a synthetic combined crRNA and tracrRNA construct called a single-guide RNA (sgRNA) ( 28 ). This was followed by studies demonstrating successful genome editing by CRISPR/Cas9 in mammalian cells, thereby opening the possibility of implementing CRISPR/Cas9 in gene therapy ( 29) ( Figure 1 ).
CRISPR- induced DSBs often trigger apoptosis rather than the intended gene edit ( 68 ). Further safety concerns were revealed when using this tool in human pluripotent stem cells (hPSCs) which demonstrated that p53 activation in response to the toxic DSBs introduced by CRISPR often triggers subsequent apoptosis ( 69 ). Thus, successful CRISPR edits are more likely to occur in p53 suppressed cells, resulting in a bias toward selection for oncogenic cell survival ( 70 ). In addition, large deletions spanning kilobases and complex rearrangements as unintended consequences of on-target activity have been reported in several instances ( 71, 72 ), highlighting a major safety issue for clinical applications of DSB-inducing CRISPR therapy. Other variations of Cas9, such as catalytically inactive endonuclease dead Cas9 (dCas9) in which the nuclease domains are deactivated, may provide therapeutic utility while mitigating the risks of DSBs ( 73 ). dCas9 can transiently manipulate expression of specific genes without introducing DSBs through fusion of transcriptional activating or repressing domains or proteins to the DNA-binding effector ( 74 ). Other variants such as Cas9n can also be considered, which induces SSBs rather than DSBs. Further modifications of these Cas9 variants has led to the development of base editors and prime editors, a key innovation for safe therapeutic application of CRISPR technology (see Precision Gene Editing With CRISPR section).
CRISPR/Cas9 is a simple two-component system used for effective targeted gene editing. The first component is the single-effector Cas9 protein, which contains the endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complementary to the spacer sequence and HNH cleaves the complementary strand. Together, these domains generate double-stranded breaks (DSBs) in the target DNA. The second component of effective targeted gene editing is a single guide RNA (sgRNA) carrying a scaffold sequence which enables its anchoring to Cas9 and a 20 base pair spacer sequence complementary to the target gene and adjacent to the PAM sequence. This sgRNA guides the CRISPR/Cas9 complex to its intended genomic location. The editing system then relies on either of two endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) ( Figure 2 ). NHEJ occurs much more frequently in most cell types and involves random insertion and deletion of base pairs, or indels, at the cut site. This error-prone mechanism usually results in frameshift mutations, often creating a premature stop codon and/or a non-functional polypeptide. This pathway has been particularly useful in genetic knock-out experiments and functional genomic CRISPR screens, but it can also be useful in the clinic in the context where gene disruption provides a therapeutic opportunity. The other pathway, which is especially appealing to exploit for clinical purposes, is the error-free HDR pathway. This pathway involves using the homologous region of the unedited DNA strand as a template to correct the damaged DNA, resulting in error-free repair. Experimentally, this pathway can be exploited by providing an exogenous donor template with the CRISPR/Cas9 machinery to facilitate the desired edit into the genome ( 30 ).
Examples of serious false-positives: 1 May 2007: a faulty virus signature issued by Symantec mistakenly removed essential operating system files, leaving thousands of PCs unable to boot. 2 May 2007: the executable file required by Pegasus Mail on Windows was falsely detected by Norton AntiVirus as being a Trojan and it was automatically removed, preventing Pegasus Mail from running. Norton AntiVirus had falsely identified three releases of Pegasus Mail as malware, and would delete the Pegasus Mail installer file when that happened. In response to this Pegasus Mail stated:
Antivirus software, or anti-virus software (abbreviated to AV software ), also known as anti-malware, is a computer program used to prevent, detect, and remove malware . Antivirus software was originally developed to detect and remove computer viruses, hence the name.
In 2007, AV-TEST reported a number of 5,490,960 new unique malware samples (based on MD5) only for that year. In 2012 and 2013, antivirus firms reported a new malware samples range from 300,000 to over 500,000 per day.
1990–2000 period (emergence of the antivirus industry) In 1990, in Spain, Mikel Urizarbarrena founded Panda Security ( Panda Software at the time). In Hungary, the security researcher Péter Szőr released the first version of Pasteur antivirus. In Italy, Gianfranco Tonello created the first version of VirIT eXplorer antivirus, ...
Anti-virus software can attempt to scan for rootkits. A rootkit is a type of malware designed to gain administrative-level control over a computer system without being detected. Rootkits can change how the operating system functions and in some cases can tamper with the anti-virus program and render it ineffective. Rootkits are also difficult to remove, in some cases requiring a complete re-installation of the operating system.
Real-time protection, on-access scanning, background guard, resident shield, autoprotect, and other synonyms refer to the automatic protection provided by most antivirus, anti-spyware, and other anti-malware programs. This monitors computer systems for suspicious activity such as computer viruses, spyware, adware, and other malicious objects. Real-time protection detects threats in opened files and scans apps in real-time as they are installed on the device. When inserting a CD, opening an email, or browsing the web, or when a file already on the computer is opened or executed.
In 1991 , in the United States, Symantec released the first version of Norton AntiVirus. In the same year, in the Czech Republic, Jan Gritzbach and Tomáš Hofer founded AVG Technologies ( Grisoft at the time), although they released the first version of their Anti-Virus Guard (AVG) only in 1992.
This means that pieces of the file, or fragments, can exist in multiple places, so when you open a file, the processor (CPU) has to retrieve all the individual fragments from different sections of the hard drive. If opening a file is taking longer than usual, disk defragmentation may help.
It is possible that your computer is running slowly because one of your components is failing. Whether it is your hard drive, RAM, CPU, or some other component, there are some cases where you may be able to replace or upgrade. However, it is likely that the best course of action is buying a new computer.
Shutting down or rebooting your computer clears out the memory cache, which will usually result in a faster, more responsive computer. If it has been a few days since you last powered down completely, it might be a good idea to give your computer a little break.
That is why your computer has a fan - to cool down the processor. If your processor is overheating , check to make sure the fan is not obstructed , that it is clean and free of debris, and that it is functioning properly.
High-content imaging (HCI) is a high-resolution platform originally developed for automated imaging and analysis of 2D cell–based assays. Newer confocal HCI instruments, designed with 3D cell models in mind, have the potential to extract high-resolution data, akin to clinical histology.
Nonuniform fluorescent labeling is a common issue for 3D models and often results from uneven diffusion of the fluorescent probe into thick 3D samples. Fix-and-stain protocols originally developed for 2D cell culture may require substantial adaptation for 3D models. For example, permeabilization and staining steps for 3D models may need to be carried out over a more extended time frame and at higher temperatures and require addition of harsher detergents and solvents to sufficiently “open up” the tissues. In addition, selecting the optimal probe concentration requires careful titration to avoid a binding-site barrier effect or nonspecific background.
Light is attenuated as it passes through dense 3D models, until it reaches a depth where the emitted light is no longer sufficient to produce a high-contrast image. Therefore, size limits must be considered and balanced with downstream analysis requirements.
3D image analysis enables the creation of 3D models which more accurately represent the true shape, location, and relationships between cells. In addition to intensity and morphological measures, the analysis software may use local intensity variation, known as texture, to further describe features of cells or subcellular components. The increased information allows for a more physiologically relevant measure of phenotypical responses from the sampled tissues.
By virtue of their more physiologically relevant architecture and cell-cell interactions, 3D cell culture models are proving to be a better alternative to 2 D cell culture models for predicting the efficacy and safety of drugs in human patients.
Later assessments of the damage showed the aftermath to be minimal. John McAfee had been quoted by the media as saying that 5 million computers would be affected. He later said that pressed by the interviewer to come up with a number, he had estimated a range from 5 thousand to 5 million, but the media naturally went with just the higher number.
Mark Washburn, working on an analysis of the Vienna and Cascade viruses with Ralf Burger, develops the first family of polymorphic viruses, the Chameleon family. Chameleon series debuted with the release of 1260. June: The Form computer virus is isolated in Switzerland.
In fiction, the 1973 Michael Crichton movie Westworld made an early mention of the concept of a computer virus, being a central plot theme that causes androids to run amok. Alan Oppenheimer 's character summarizes the problem by stating that "...there's a clear pattern here which suggests an analogy to an infectious disease process, spreading from one...area to the next." To which the replies are stated: "Perhaps there are superficial similarities to disease" and, "I must confess I find it difficult to believe in a disease of machinery." (Crichton's earlier work, the 1969 novel The Andromeda Strain and 1971 film were about an extraterrestrial biological virus-like disease that threatened the human race.)
November: The term "virus" is re-coined by Frederick B. Cohen in describing self-replicating computer programs. In 1984 Cohen uses the phrase "computer virus" (suggested by his teacher Leonard Adleman) to describe the operation of such programs in terms of "infection". He defines a "virus" as "a program that can 'infect' other programs by modifying them to include a possibly evolved copy of itself." Cohen demonstrates a virus-like program on a VAX11/750 system at Lehigh University. The program could install itself in, or infect, other system objects.
A program called Elk Cloner, written for Apple II systems, was created by high school student Richard Skrenta, originally as a prank. The Apple II was particularly vulnerable due to the storage of its operating system on a floppy disk. Elk Cloner's design combined with public ignorance about what malware was and how to protect against it led to Elk Cloner being responsible for the first large-scale computer virus outbreak in history.
February 16: The Netsky worm is discovered. The worm spreads by email and by copying itself to folders on the local hard drive as well as on mapped network drives if available.
The rootkit creates vulnerabilities on affected computers, making them susceptible to infection by worms and viruses. Late 2005: The Zlob Trojan, is a Trojan horse program that masquerades as a required video codec in the form of the Microsoft Windows ActiveX component. It was first detected in late 2005.