Cancer Biology and Genetics for Non-Biologists
Trevor F. Cox in Medical Statistics for Cancer Studies, 2022
All living things are made up of cells, from the simple unicellular amoeba to the complex human composed of about 37 trillion () cells. Cells that contain a nucleus are called eukaryotic cells; cells without a nucleus are called prokaryotic cells. Bacteria are examples of prokaryotic cells. Humans are eukaryotes consisting of eukaryotic cells, such as bone, nerve and stem cells. In fact, there are about 200 types of cells in our bodies. Figure 2.1 shows a typical eukaryotic cell, illustrating its structure. Cells come in different shapes and sizes; neurons in the brain and nervous system are long and thin, blood cells are roughly spherical, some bone cells are cuboidal and columnar while others have many branches. The size of a red blood cell is , the size of a skin cell is , an ovum , whilst the length of some nerve cells can be over .
Potential of Antibody Therapy for Respiratory Virus Infections
Sunit K. Singh in Human Respiratory Viral Infections, 2014
Offering one of the largest libraries of up to 1014 clones, ribosome or mRNA display can be performed in both eukaryotic and prokaryotic cells [53,59]. The process involves the formation of a stable complex of mRNA and antibody (or fragment) in a similar concept of fragment display. In ribosome display, a ribosome that has been halted by chloramphenicol or cycloheximide (in prokaryotic or eukaryotic cells, respectively) is also linked to the stable complex. In mRNA display, the antibody fragment is linked to the mRNA via a puromycin linker [53,60]. Thereafter, it follows the same screening concepts as phage display, with the use of a mild eluting step (EDTA) to destabilize the mRNA and the addition of an RT-PCR amplifying step [53,61]. Ribosome display may prove more challenging to accomplish compared to traditional phage display due to RNA or ribosomal instability, and is limited to single-chain formats such as scFv [62].
Laboratory techniques to study the cellular and molecular processes of disorders
Louis-Philippe Boulet in Applied Respiratory Pathophysiology, 2017
Clustered regularly interspaced short palindromic repeats (CRISPR) refers to the natural occurrence of a DNA repeating sequence separated by a nonrepeating sequence, called spacer, in the prokaryotic genomes [50] (Figure 3.11). It is a defense mechanism used by prokaryotes to fight off viral infection [51,52]. Briefly, a spacer corresponding to a previously exposed viral sequence is kept between repeating DNA sequences so that it can be used to recognize the same virus in the future. To recognize and defend against a viral infection, the prokaryote transcribes the sequence into an RNA strand and together with a Cas enzyme, the complex drifts around inside the cell. Once a viral sequence matches the RNA sequence, the Cas enzyme snips the viral DNA and prevents the virus from replicating. Of the various Cas enzymes, Cas9 is the best known and it comes from Streptococcus pyogenes [53]. The derived gene-editing system is thus known as the CRISPR-Cas9 system. Briefly, this gene-editing method begins with a Cas9 enzyme guided to a specific DNA site with the help of a customized 17–20-nucleotide long guide RNA (gRNA). Cas9 snips the targeted DNA at the precise location generating a double-strand break (DSB) [54]. The cleaved DSB can be repaired by an error prone nonhomologous end joining (NHEJ) mechanism and generates random microinsertion or microdeletion (INDEL) mutations, or it can be replaced with a gene modification by homology-directed repair (HDR) in the presence of donor template [55]. Hence, NHEJ and HDR can be adapted to either silence a gene or replace a gene, respectively [56].
A review of co-culture models to study the oral microenvironment and disease
Published in Journal of Oral Microbiology, 2020
Sophie E Mountcastle, Sophie C Cox, Rachel L Sammons, Sara Jabbari, Richard M Shelton, Sarah A Kuehne
Co-culture techniques allow a variety of cell types to be cultivated together, enabling examination of cell–cell interactions [10]. These systems may refer to the culture of two or more eukaryotic cell types together, or eukaryotic and prokaryotic cells. The effectiveness of co-cultures is heavily determined by the choice of experimental setup. Cell–cell interactions in co-cultures are strongly influenced by the extracellular environment, which in turn is influenced by the employed protocol [11]. There are numerous factors that need to be optimised to ensure these systems are representative of the native oral cavity, such as the number of cell populations. Having more than two species can result in unstable systems due to multiple reaction pathways, which may be difficult to monitor, analyse, and interpret [11].
Higher accuracy of genotypic identification compared to phenotyping in the diagnosis of coagulase-negative staphylococcus infection in orthopedic surgery
Published in Infectious Diseases, 2020
Gema Muñoz-Gamito, Eva Cuchí, Jordi Roigé, Lucía Gómez, Àngels Jaén, Alfredo Matamala, M. Lluïsa Pedro-Botet, Josep Anton Capdevila, Francesc Anglès, Josefa Pérez
Microbial identification based on genotyping has proven to be more accurate than phenotyping in other clinical scenarios, such as catheter-related infections [2], but the usefulness of this technique has not been investigated in bone and joint infection. Repetitive extragenic palindromic PCR (rep-PCR) is a technique based on amplification and detection of the repetitive DNA characteristically found in microorganisms. These sequences can be used to construct primers that amplify the regions located between consecutive sequences in a genome. In prokaryotic cells, these sequences are short (<200 bp), widely distributed along the entire genome, and do not code for proteins. One of the first sequences described and studied was named REP (repetitive extragenic palindrome). This term has been used to name the technique. Genetic variants in these sequences produce characteristic amplification patterns that can be identified as DNA bands in electrophoresis gels. With the use of rep-PCR, two strains can be identified as being identical with 97% certainty. For this reason, it is the current reference method for identifying virulent strains in nosocomial and community outbreaks [3–5].
Gene editing technology: Towards precision medicine in inherited retinal diseases
Published in Seminars in Ophthalmology, 2021
Brian G. Ballios, Eric A. Pierce, Rachel M. Huckfeldt
In the late 1980s, it was discovered that the E. coli genome harbored clustered regularly interspaced short palindromic repeats (CRISPR), 81 which were later found to harbor sequences that match viral genomes.82,83 CRISPR-associated (Cas) proteins were discovered that can capture or cleave, and inactivate, the genome of invading viruses, guided by short RNA (crRNA) sequences84 coupled with trans-acting RNA (tracrRNA) that participates in processing and cleavage of the invading DNA.85 CRISPR gives these prokaryotes the ability to recognize the genetic sequence of an invading DNA-virus and target it for destruction. This constitutes what can be imagined as a prokaryote “immune system” to defend against infection from invading viruses. These three components (Cas, crRNA, and tracrRNA) form the basic structure of the endonuclease that is now used for gene editing,86 and it was not long after its discovery that it was shown that modified CRISPR-Cas9 could be used to make targeted edits in the mouse and human genome.87
Related Knowledge Centers
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