RNA interference

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translation or transcriptional repression. Historically, RNAi was known by other names, including "co-suppression", "post-transcriptional gene silencing" (PTGS), and "quelling". The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm "Caenorhabditis elegans", which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.
Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can direct enzyme complexes to degrade messenger RNA (mRNA) molecules and thus decrease their activity by preventing translation, via post-transcriptional gene silencing. Moreover, transcription can be inhibited via the pre-transcriptional silencing mechanism of RNA interference, through which an enzyme complex catalyzes DNA methylation at genomic positions complementary to complexed siRNA or miRNA. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.
The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded fragments of ~21 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute 2 (Ago2), the catalytic component of the RISC. In some organisms, this process spreads systemically, despite the initially limited molar concentrations of siRNA.
RNAi is a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can selectively and robustly induce suppression of specific genes of interest. RNAi may be used for large-scale screens that systematically shut down each gene in the cell, which can help to identify the components necessary for a particular cellular process or an event such as cell division. The pathway is also used as a practical tool in biotechnology, medicine and insecticides.
Cellular mechanism.
RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute. When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by Dicer. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC.
Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves double-stranded RNAs (dsRNAs) in plants, or short hairpin RNAs (shRNAs) in humans, to produce double-stranded fragments of 20–25 base pairs with a 2-nucleotide overhang at the 3' end. Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects. These short double-stranded fragments are called small interfering RNAs (siRNAs). These siRNAs are then separated into single strands and integrated into an active RISC, by RISC-Loading Complex (RLC). RLC includes Dicer-2 and R2D2, and is crucial to unite Ago2 and RISC. TATA-binding protein-associated factor 11 (TAF11) assembles the RLC by facilitating Dcr-2-R2D2 tetramerization, which increases the binding affinity to siRNA by 10-fold. Association with TAF11 would convert the R2-D2-Initiator (RDI) complex into the RLC. R2D2 carries tandem double-stranded RNA-binding domains to recognize the thermodynamically stable terminus of siRNA duplexes, whereas Dicer-2 the other less stable extremity. Loading is asymmetric: the MID domain of Ago2 recognizes the thermodynamically stable end of the siRNA. Therefore, the "passenger" (sense) strand whose 5′ end is discarded by MID is ejected, while the saved "guide" (antisense) strand cooperates with AGO to form the RISC.
After integration into the RISC, siRNAs base-pair to their target mRNA and cleave it, thereby preventing it from being used as a translation template. Differently from siRNA, a miRNA-loaded RISC complex scans cytoplasmic mRNAs for potential complementarity. Instead of destructive cleavage (by Ago2), miRNAs rather target the 3′ untranslated region (UTR) regions of mRNAs where they typically bind with imperfect complementarity, thus blocking the access of ribosomes for translation.
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in "C. elegans" and R2D2 in "Drosophila", that stimulates dicer activity. The mechanism producing this length specificity is unknown and this protein only binds long dsRNAs.
In "C. elegans" this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the dicer-produced initiating or 'primary' siRNAs are used as templates. These 'secondary' siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).
MicroRNA.
MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of miRNAs as well as silencing triggered by foreign dsRNA. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modification. A miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a "pri-miRNA" which is processed, in the cell nucleus, to a 70-nucleotide stem-loop structure called a "pre-miRNA" by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein DGCR8. The dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same downstream cellular machinery. First, viral encoded miRNA was described in Epstein–Barr virus (EBV). Thereafter, an increasing number of microRNAs have been described in viruses. VIRmiRNA is a comprehensive catalogue covering viral microRNA, their targets and anti-viral miRNAs (see also VIRmiRNA resource: http://crdd.osdd.net/servers/virmirna/).
siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target. In "Drosophila" and "C. elegans", miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.
Three prime untranslated regions and microRNAs.
Three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.
The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.
As of 2014, the miRBase web site, an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). Friedman et al. estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.
Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).
The effects of miRNA dysregulation of gene expression seem to be important in cancer. For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.
The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders.
RISC activation and catalysis.
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in "C. elegans" and R2D2 in "Drosophila", that stimulates dicer activity. This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown. This RNA-binding protein then facilitates the transfer of cleaved siRNAs to the RISC complex.
In "C. elegans" this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the dicer-produced initiating or 'primary' siRNAs are used as templates. These 'secondary' siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).
The active components of an RNA-induced silencing complex (RISC) are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the "guide strand", binds the argonaute protein and directs gene silencing. The other "anti-guide strand" or "passenger strand" is degraded during RISC activation. Although it was first believed that an ATP-dependent helicase separated these two strands, the process proved to be ATP-independent and performed directly by the protein components of RISC. However, an "in vitro" kinetic analysis of RNAi in the presence and absence of ATP showed that ATP may be required to unwind and remove the cleaved mRNA strand from the RISC complex after catalysis. The guide strand tends to be the one whose 5' end is less stably paired to its complement, but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation. Instead, the R2D2 protein may serve as the differentiating factor by binding the more-stable 5' end of the passenger strand.
The structural basis for binding of RNA to the argonaute protein was examined by X-ray crystallography of the binding domain of an RNA-bound argonaute protein. Here, the phosphorylated 5' end of the RNA strand enters a conserved basic surface pocket and makes contacts through a divalent cation (an atom with two positive charges) such as magnesium and by aromatic stacking (a process that allows more than one atom to share an electron by passing it back and forth) between the 5' nucleotide in the siRNA and a conserved tyrosine residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target. Analysis of the inhibitory effect of mismatches in either the 5’ or 3’ end of the guide strand has demonstrated that the 5’ end of the guide strand is likely responsible for matching and binding the target mRNA, while the 3’ end is responsible for physically arranging target mRNA into a cleavage-favorable RISC region.
It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation, translation of the mRNA target is not essential for RNAi-mediated degradation. Indeed, RNAi may be more effective against mRNA targets that are not translated. Argonaute proteins are localized to specific regions in the cytoplasm called P-bodies (also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay; miRNA activity is also clustered in P-bodies. Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are a critical site in the RNAi process.
Transcriptional silencing.
Components of the RNAi pathway are used in many eukaryotes in the maintenance of the organization and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serves to downregulate genes pre-transcriptionally; this process is referred to as RNA-induced transcriptional silencing (RITS), and is carried out by a complex of proteins called the RITS complex. In fission yeast this complex contains argonaute, a chromodomain protein Chp1, and a protein called Tas3 of unknown function. As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins. Indeed, deletion of these genes in the fission yeast "S. pombe" disrupts histone methylation and centromere formation, causing slow or stalled anaphase during cell division. In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.
The mechanism by which the RITS complex induces heterochromatin formation and organization is not well understood. Most studies have focused on the mating-type region in fission yeast, which may not be representative of activities in other genomic regions/organisms. In maintenance of existing heterochromatin regions, RITS forms a complex with siRNAs complementary to the local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that are initiated by RNA polymerase. The formation of such a heterochromatin region, though not its maintenance, is dicer-dependent, presumably because dicer is required to generate the initial complement of siRNAs that target subsequent transcripts. Heterochromatin maintenance has been suggested to function as a self-reinforcing feedback loop, as new siRNAs are formed from the occasional nascent transcripts by RdRP for incorporation into local RITS complexes. The relevance of observations from fission yeast mating-type regions and centromeres to mammals is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.
Crosstalk with RNA editing.
The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine nucleotides into inosine in dsRNAs via the enzyme adenosine deaminase (ADAR). It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate. Some pre-miRNAs do undergo A→I RNA editing and this mechanism may regulate the processing and expression of mature miRNAs. Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components. Further support for this model comes from studies on ADAR-null "C. elegans" strains indicating that A→I RNA editing may counteract RNAi silencing of endogenous genes and transgenes.
Variation among organisms.
Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and "C. elegans", although not in "Drosophila" or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata (channels in the cell walls that enable communication and transport). Heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell. A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression. This translational effect may be produced by inhibiting the interactions of translation initiation factors with the messenger RNA's polyadenine tail.
Some eukaryotic protozoa such as "Leishmania major" and "Trypanosoma cruzi" lack the RNAi pathway entirely. Most or all of the components are also missing in some fungi, most notably the model organism "Saccharomyces cerevisiae". The presence of RNAi in other budding yeast species such as "Saccharomyces castellii" and "Candida albicans", further demonstrates that inducing two RNAi-related proteins from "S. castellii" facilitates RNAi in "S. cerevisiae". That certain ascomycetes and basidiomycetes are missing RNA interference pathways indicates that proteins required for RNA silencing have been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niches.
Related prokaryotic systems.
Gene expression in prokaryotes is influenced by an RNA-based system similar in some respects to RNAi. Here, RNA-encoding genes control mRNA abundance or translation by producing a complementary RNA that anneals to an mRNA. However these regulatory RNAs are not generally considered to be analogous to miRNAs because the dicer enzyme is not involved. It has been suggested that CRISPR interference systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components are orthologous.
Biological functions.
Immunity.
RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may also prevent the self-propagation of transposons. Plants such as "Arabidopsis thaliana" express multiple dicer homologs that are specialized to react differently when the plant is exposed to different viruses. Even before the RNAi pathway was fully understood, it was known that induced gene silencing in plants could spread throughout the plant in a systemic effect and could be transferred from stock to scion plants via grafting. This phenomenon has since been recognized as a feature of the plant adaptive immune system and allows the entire plant to respond to a virus after an initial localized encounter. In response, many plant viruses have evolved elaborate mechanisms to suppress the RNAi response. These include viral proteins that bind short double-stranded RNA fragments with single-stranded overhang ends, such as those produced by dicer. Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria. These effects may be part of a generalized response to pathogens that downregulates any metabolic process in the host that aids the infection process.
Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals produces an antiviral response. In both juvenile and adult "Drosophila", RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus. A similar role in immunity may operate in "C. elegans", as argonaute proteins are upregulated in response to viruses and worms that overexpress components of the RNAi pathway are resistant to viral infection.
The role of RNA interference in mammalian innate immunity is poorly understood, and relatively little data is available. However, the existence of viruses that encode genes able to suppress the RNAi response in mammalian cells may be evidence in favour of an RNAi-dependent mammalian immune response, although this hypothesis has been challenged as poorly substantiated.
Evidence for the existence of a functional antiviral RNAi pathway in mammalian cells has been presented.
Other functions for RNAi in mammalian viruses also exist, such as miRNAs expressed by the herpes virus that may act as heterochromatin organization triggers to mediate viral latency.
Downregulation of genes.
Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most important in translational repression and in the regulation of development, especially on the timing of morphogenesis and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cells. The role of endogenously expressed miRNA in downregulating gene expression was first described in "C. elegans" in 1993. In plants this function was discovered when the "JAW microRNA" of "Arabidopsis" was shown to be involved in the regulation of several genes that control plant shape. In plants, the majority of genes regulated by miRNAs are transcription factors; thus miRNA activity is particularly wide-ranging and regulates entire gene networks during development by modulating the expression of key regulatory genes, including transcription factors as well as F-box proteins. In many organisms, including humans, miRNAs are linked to the formation of tumors and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumor suppressors.
Evolution.
Based on parsimony-based phylogenetic analysis, the most recent common ancestor of all eukaryotes most likely already possessed an early RNA interference pathway; the absence of the pathway in certain eukaryotes is thought to be a derived characteristic. This ancestral RNAi system probably contained at least one dicer-like protein, one argonaute, one PIWI protein, and an RNA-dependent RNA polymerase that may also have played other cellular roles. A large-scale comparative genomics study likewise indicates that the eukaryotic crown group already possessed these components, which may then have had closer functional associations with generalized RNA degradation systems such as the exosome. This study also suggests that the RNA-binding argonaute protein family, which is shared among eukaryotes, most archaea, and at least some bacteria (such as "Aquifex aeolicus"), is homologous to and originally evolved from components of the translation initiation system.
Applications.
Gene knockdown.
The RNA interference pathway is often exploited in experimental biology to study the function of genes in cell culture and "in vivo" in model organisms. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated. In a recent study validation of RNAi silencing efficiency using gene array data showed 18.5% failure rate across 429 independent experiments.
Extensive efforts in computational biology have been directed toward the design of successful dsRNA reagents that maximize gene knockdown but minimize "off-target" effects. Off-target effects arise when an introduced RNA has a base sequence that can pair with and thus reduce the expression of multiple genes. Such problems occur more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of humans, "C. elegans" and "S. pombe" that about 10% of possible siRNAs have substantial off-target effects. A multitude of software tools have been developed implementing algorithms for the design of general mammal-specific, and virus-specific siRNAs that are automatically checked for possible cross-reactivity.
Depending on the organism and experimental system, the exogenous RNA may be a long strand designed to be cleaved by dicer, or short RNAs designed to serve as siRNA substrates. In most mammalian cells, shorter RNAs are used because long double-stranded RNA molecules induce the mammalian interferon response, a form of innate immunity that reacts nonspecifically to foreign genetic material. Mouse oocytes and cells from early mouse embryos lack this reaction to exogenous dsRNA and are therefore a common model system for studying mammalian gene-knockdown effects. Specialized laboratory techniques have also been developed to improve the utility of RNAi in mammalian systems by avoiding the direct introduction of siRNA, for example, by stable transfection with a plasmid encoding the appropriate sequence from which siRNAs can be transcribed, or by more elaborate lentiviral vector systems allowing the inducible activation or deactivation of transcription, known as "conditional RNAi".
Functional genomics.
Approaches to the design of genome-wide RNAi libraries can require more sophistication than the design of a single siRNA for a defined set of experimental conditions. Artificial neural networks are frequently used to design siRNA libraries and to predict their likely efficiency at gene knockdown. Mass genomic screening is widely seen as a promising method for genome annotation and has triggered the development of high-throughput screening methods based on microarrays. However, the utility of these screens and the ability of techniques developed on model organisms to generalize to even closely related species has been questioned, for example from "C. elegans" to related parasitic nematodes.
Medicine.
History of RNAi use in medicine
The first instance of RNA silencing in animals was documented in 1996, when Guo and Kemphues observed that, by introducing sense and antisense RNA to par-1 mRNA in "Caenorhabditis elegans" caused degradation of the par-1 message. It was thought that this degradation was triggered by single-stranded RNA (ssRNA), but two years later, in 1998, Fire and Mello discovered that this ability to silence the par-1 gene expression was actually triggered by double-stranded RNA (dsRNA). They would eventually share the Nobel Prize in Physiology or Medicine for this discovery. Just after Fire and Mello's ground-breaking discovery, Elbashir et al. discovered, by using synthetically made small interfering RNA (siRNA), it was possible to target the silencing of specific sequences in a gene, rather than silencing the entire gene. Only a year later, McCaffrey and colleagues demonstrated that this sequence-specific silencing had therapeutic applications by targeting a sequence from the Hepatitis C virus in transgenic mice. Since then, multiple researchers have been attempting to expand the therapeutic applications of RNAi, specifically looking to target genes that cause various types of cancer. By 2006, the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus. Four years later the first-in-human Phase I clinical trial was started, using a nanoparticle delivery system to target solid tumors. Although most research is currently looking into the applications of RNAi in cancer treatment, the list of possible applications is extensive. RNAi could potentially be used to treat viruses, bacterial diseases, parasites, maladaptive genetic mutations, control drug consumption, provide pain relief, and even modulate sleep.
Therapeutic applications.
Viral infection.
Antiviral treatment is one of the earliest proposed RNAi-based medical applications, and two different types have been developed. The first type is to target viral RNAs. Many studies have shown that targeting viral RNAs can suppress the replication of numerous viruses, including HIV, HPV, hepatitis A, hepatitis B, influenza virus, respiratory syncytial virus (RSV), SARS coronavirus (SARS-CoV), adenovirus and measles virus. The other strategy is to block the initial viral entries by targeting the host cell genes. For example, suppression of chemokine receptors (CXCR4 and CCR5) on host cells can prevent HIV viral entry.
Cancer.
While traditional chemotherapy can effectively kill cancer cells, lack of specificity for discriminating normal cells and cancer cells in these treatments usually cause severe side effects. Numerous studies have demonstrated that RNAi can provide a more specific approach to inhibit tumor growth by targeting cancer-related genes (i.e., oncogene). It has also been proposed that RNAi can enhance the sensitivity of cancer cells to chemotherapeutic agents, providing a combinatorial therapeutic approach with chemotherapy. Another potential RNAi-based treatment is to inhibit cell invasion and migration.
Neurological diseases.
RNAi strategies also show potential for treating neurodegenerative diseases. Studies in cells and in mouse have shown that specifically targeting Amyloid beta-producing genes (e.g. BACE1 and APP) by RNAi can significantly reduced the amount of Aβ peptide which is correlated with the cause of Alzheimer's disease. In addition, this silencing-based approaches also provide promising results in treatment of Parkinson's disease and Polyglutamine disease.
Difficulties in therapeutic application.
To achieve the clinical potential of RNAi, siRNA must be efficiently transportated to the cells of target tissues. However, there are various barriers that must be fixed before it can be used clinically. For example, "naked" siRNA is susceptible to several obstacles that reduce its therapeutic efficacy. Additionally, once siRNA has entered the bloodstream, naked RNA can be degraded by serum nucleases and can stimulate the innate immune system. Due to its size and highly polyanionic (containing negative charges at several sites) nature, unmodified siRNA molecules cannot readily enter the cells through the cell membrane. Therefore, artificial or nanoparticle encapsulated siRNA must be used. However, transporting siRNA across the cell membrane still has its own unique challenges. If siRNA is transferred across the cell membrane, unintended toxicities can occur if therapeutic doses are not optimized, and siRNAs can exhibit off-target effects (e.g. unintended downregulation of genes with partial sequence complementarity). Even after entering the cells, repeated dosing is required since their effects are diluted at each cell division. As previously described, parts of the vector that transports dsRNA can also have regulatory effects. Therefore, unspecific side effects should be considered and controlled.
Cancer treatment.
Compared with chemotherapy or other anti-cancer drugs, there are a lot of advantages of siRNA drug. SiRNA acts on the post-transcriptional stage of gene expression, so it does not modify or change DNA in a deleterious effect. SiRNA can also be used to produce a specific response in a certain type of way, such as by downgrading suppression of gene expression. In a single cancer cell, siRNA can cause dramatic suppression of gene expression with just several copies. This happens by silencing cancer-promoting genes with RNAi, as well as targeting an mRNA sequence.
RNAi drugs treat cancer by silencing certain cancer promoting genes. This is done by complementing the cancer genes with the RNAi, such as keeping the mRNA sequences in accordance with the RNAi drug. Ideally, RNAi is should be injected and/or chemically modified so the RNAi can reach cancer cells more efficiently. RNAi uptake and regulation is monitored by the kidneys.
Stimulation of immune response.
The human immune system is divided into two separate branches: the innate immune system and the adaptive immune system. The innate immune system is the first defense against infection and responds to pathogens in a generic fashion. On the other hand, the adaptive immune system, a system that was evolved later than the innate, is composed mainly of highly specialized B and T cells that are trained to react to specific portions of pathogenic molecules.
The challenge between old pathogens and new has helped create a system of guarded cells and particles that are called safe framework. This framework has given humans an army systems that search out and destroy invader particles, such as pathogens, microscopic organisms, parasites, and infections. The mammalian safe framework has developed to incorporate siRNA as a tool to indicate viral contamination, which has allowed siRNA is create an intense innate immune response.
siRNA is controlled by the innate immune system, which can be divided into the acute inflammatory responses and antiviral responses. The inflammatory response is created with signals from small signaling molecules, or cytokines. These include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor α (TNF-α). The innate immune system generates inflammation and antiviral responses, which cause the release pattern recognition receptors (PRRs). These receptors help in labeling which pathogens are viruses, fungi, or bacteria. Moreover, the importance of siRNA and the innate immune system is to include more PRRs to help recognize different RNA structures. This makes it more likely for the siRNA to cause an immunostimulant response in the event of the pathogen.
Prospects as a therapeutic technique.
Clinical Phase I and II studies of siRNA therapies conducted between 2015 and 2017 have demonstrated potent and durable gene knockdown in the liver, with some signs of clinical improvement and without unacceptable toxicity. Two Phase III studies are in progress to treat familial neurodegenerative and cardiac syndromes caused by mutations in transthyretin (TTR). Numerous publications have shown that in vivo delivery systems are very promising and are diverse in characteristics, allowing numerous applications. The nanoparticle delivery system shows the most promise yet this method presents additional challenges in the scale-up of the manufacturing process, such as the need for tightly controlled mixing processes to achieve consistent quality of the drug product.
The table below shows different drugs using RNA interference and what their phases and status was in clinical trials as of 2013.
Biotechnology.
RNA interference has been used for applications in biotechnology and is nearing commercialization in other fields. RNAi has resulted in the invention of novel crops such as nicotine-free tobacco, decaffeinated coffee, nutrient fortified vegetation, and hypoallergenic crops. The genetically-engineered Arctic apples received FDA approval in 2015. The apples were produced by RNAi suppression of the PPO (polyphenol oxidase) gene, making apple varieties that will not undergo browning after being sliced. PPO-silenced apples are unable to convert chlorogenic acid into the standard quinone product.
There are several opportunities for the applications of RNAi in crop science for its improvement such as stress tolerance and enhanced nutritional level. RNAi will prove its potential for inhibition of photorespiration to enhance the productivity of C3 plants. This knockdown technology may be useful in inducing early flowering, delayed ripening, delayed senescence, breaking dormancy, stress-free plants, overcoming self-sterility, etc.
Foods.
RNAi has been used to genetically engineer plants to produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. Cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is itself important in preventing damage from plant pests. Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.
No plant products that use RNAi-based genetic engineering have yet exited the experimental stage. Development efforts have successfully reduced the levels of allergens in tomato plants and fortification of plants such as tomatoes with dietary antioxidants. Previous commercial products, including the Flavr Savr tomato and two cultivars of ringspot-resistant papaya, were originally developed using antisense technology but likely exploited the RNAi pathway. RNAi silencing of alpha-amylase have also been used to decrease Aspergillus flavus fungal growth in maize which would have otherwise contaminated the kernels with dangerous aflatoxins. Silencing lachrymatory factor synthase in onions have produced tearless onions and RNAi has been used in BP1 genes in rapeseeds to improve photosynthesis. SBEIIa and SBEIIb genes in wheat have been targeted in wheat in order to produce higher levels of amylose in order to improve bowel function.
Other crops.
Another effort decreased the precursors of likely carcinogens in tobacco plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy and resistance to common plant viruses.
Insecticide.
RNAi is under development as an insecticide, employing multiple approaches, including genetic engineering and topical application. Cells in the midgut of some insects take up the dsRNA molecules in the process referred to as environmental RNAi. In some insects the effect is systemic as the signal spreads throughout the insect's body (referred to as systemic RNAi).
Animals exposed to RNAi at doses millions of times higher than anticipated human exposure levels show no adverse effects.
RNAi has varying effects in different species of Lepidoptera (butterflies and moths). Possibly because their saliva and gut juice is better at breaking down RNA, the cotton bollworm, the beet armyworm and the Asiatic rice borer have so far not been proven susceptible to RNAi by feeding.
Recent evidence suggests that resistance to RNAi could be broad-spectrum, meaning that resistance to one sequence could confer resistance to other dsRNA sequences. In one laboratory population of western corn rootworm, resistance occurred through lack of uptake of DvSnf7 dsRNA through the gut. When other dsRNA sequences were tested against DvSnf7, the other sequences were no longer effective which suggests that resistance management would be more difficult than simply switching out dsRNA sequences. Combining multiple strategies, such as engineering the protein Cry, derived from a bacterium called "Bacillus thuringiensis" (Bt), and RNAi in one plant delay the onset of resistance.
Transgenic plants.
Transgenic crops have been made to express dsRNA, carefully chosen to silence crucial genes in target pests. These dsRNAs are designed to affect only insects that express specific gene sequences. As a proof of principle, in 2009 a study showed RNAs that could kill any one of four fruit fly species while not harming the other three.
In 2012 Syngenta bought Belgian RNAi firm Devgen for $522 million and Monsanto paid $29.2 million for the exclusive rights to intellectual property from Alnylam Pharmaceuticals. The International Potato Center in Lima, Peru is looking for genes to target in the sweet potato weevil, a beetle whose larvae ravage sweet potatoes globally. Other researchers are trying to silence genes in ants, caterpillars and pollen beetles. Monsanto will likely be first to market, with a transgenic corn seed that expresses dsRNA based on gene Snf7 from the western corn rootworm, a beetle whose larvae annually cause one billion dollars in damage in the United States alone. A 2012 paper showed that silencing Snf7 stunts larval growth, killing them within days. In 2013 the same team showed that the RNA affects very few other species.
Topical.
Alternatively dsRNA can be supplied without genetic engineering. One approach is to add them to irrigation water. The molecules are absorbed into the plants' vascular system and poison insects feeding on them. Another approach involves spraying dsRNA like a conventional pesticide. This would allow faster adaptation to resistance. Such approaches would require low cost sources of dsRNAs that do not currently exist.
Genome-scale screening.
Genome-scale RNAi research relies on high-throughput screening (HTS) technology. RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific phenotypes. This technology has been hailed as a potential second genomics wave, following the first genomics wave of gene expression microarray and single nucleotide polymorphism discovery platforms.
One major advantage of genome-scale RNAi screening is its ability to simultaneously interrogate thousands of genes. With the ability to generate a large amount of data per experiment, genome-scale RNAi screening has led to an explosion of data generation rates. Exploiting such large data sets is a fundamental challenge, requiring suitable statistics/bioinformatics methods. The basic process of cell-based RNAi screening includes the choice of an RNAi library, robust and stable cell types, transfection with RNAi agents, treatment/incubation, signal detection, analysis and identification of important genes or therapeutical targets.
History.
The process of RNAi was referred to as "co-suppression" and "quelling" when observed prior to the knowledge of an RNA-related mechanism. The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants, and more directly by reports of unexpected outcomes in experiments performed by plant scientists in the United States and the Netherlands in the early 1990s. In an attempt to alter flower colors in petunias, researchers introduced additional copies of a gene encoding chalcone synthase, a key enzyme for flower pigmentation into petunia plants of normally pink or violet flower color. The overexpressed gene was expected to result in darker flowers, but instead caused some flowers to have less visible purple pigment, sometimes in variegated patterns, indicating that the activity of chalcone synthase had been substantially decreased or became suppressed in a context-specific manner. This would later be explained as the result of the transgene being inserted adjacent to promoters in the opposite direction in various positions throughout the genomes of some transformants, thus leading to expression of antisense transcripts and gene silencing when these promoters are active. Another early observation of RNAi came from a study of the fungus "Neurospora crassa", although it was not immediately recognized as related. Further investigation of the phenomenon in plants indicated that the downregulation was due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation. This phenomenon was called "co-suppression of gene expression", but the molecular mechanism remained unknown.
Not long after, plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon. While it was known that plants expressing virus-specific proteins showed enhanced tolerance or resistance to viral infection, it was not expected that plants carrying only short, non-coding regions of viral RNA sequences would show similar levels of protection. Researchers believed that viral RNA produced by transgenes could also inhibit viral replication. The reverse experiment, in which short sequences of plant genes were introduced into viruses, showed that the targeted gene was suppressed in an infected plant. This phenomenon was labeled "virus-induced gene silencing" (VIGS), and the set of such phenomena were collectively called "post transcriptional gene silencing."
After these initial observations in plants, laboratories searched for this phenomenon in other organisms. Craig C. Mello and Andrew Fire's 1998 "Nature" paper reported a potent gene silencing effect after injecting double stranded RNA into "C. elegans". In investigating the regulation of muscle protein production, they observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term "RNAi". This discovery represented the first identification of the causative agent for the phenomenon. Fire and Mello were awarded the 2006 Nobel Prize in Physiology or Medicine.


Armillaria nabsnona

Armillaria nabsnona is a species of fungus in the family Physalacriaceae. The species is found in the west coast of North America, Hawaii, and Japan, where it grows on decaying hardwoods, particularly species of alder. Its fruit bodies have convex to flattened orange-brown caps up to in diameter, brown stipes, and whitish to pinkish-tan gills.
Taxonomy.
The species was described scientifically in 1996 by American mycologists Tom Volk and Harold Burdsall. Although the existence of the species as a distinct taxon was known for several years before that, it took several years to collect sufficient specimens and determine an appropriate type collection. The original published description is based on collections found growing on the fallen trunk of "Acer macrophyllum" in Olympic National Park, Washington, in October 1993. The specific epithet "nabsnona" is derived from an acronym of "North American Biological Specimen" and "nona" (ninth).
Description.
The fruit bodies have caps that are initially convex before flattening in age, and reach diameters between . The cap has a smooth surface that becomes sticky when wet. Young specimens can have short, dark hairs on the center. The cap color is brownish, becoming paler towards the edge, and frequently has darker, irregularly shaped bruise spots. The cap margin appears grooved or furrowed due to the thin flesh (measuring 0.5–1 mm thick) and the gills underneath. The gills have an adnate to somewhat decurrent attachment to the stipe. They are somewhat distantly spaced, 0.75–1 mm wide, and initially white to cream before darkening to pinkish-tan in maturity. The brownish stipe measures long by 2–3 mm thick, with a broader base measuring 4–5 mm. It has a ring, and whitish patches of cottony mycelium on its surface below the ring. The ring develops from a dense, white and cottony partial veil that becomes ragged as the cap grows, and sometimes persists as a temporary cortina. Rhizomorphs, if present, are black, branched, and 1–2 mm thick.
The spore print is white. Spores are smooth, hyaline (translucent), inamyloid, and have an ovoid to somewhat more or less spherical shape. They typically measure in the range 8–10 by 5.5–6.5 µm. The basidia are club-shaped, four-spored, and 25–35 by 5.5–6 µm, and have a clamp at their bases. Additional basidia may arise from the basal clamp, a branching pattern that distinguishes this species from other "Armillaria". This feature becomes less visually prominent as the hymenium matures and neighboring basidia expand and become crowded. The mycelium of the fungus is bioluminescent.
Habitat and distribution.
The fruit bodies of "Armillaria nabsnona" grow in groups, but not clustered together at the base. They are found on hardwood trees (especially "Alnus") in riparian areas. Although it fruits most commonly in autumn, it has been recorded fruiting in the spring in Oregon The range of the fungus was originally described as covering roughly the Pacific Northwest region of North America, including the US states of Washington, Oregon, California, Idaho, Alaska, and the Canadian province British Columbia. In 2008 it was recorded in Hawaii; in 2009, it was reported from Hokkaido Island in northern Japan. Japanese researchers have reported a symbiotic association with the orchid "Gastrodia elata".


Paeniglutamicibacter antarcticus

Paeniglutamicibacter antarcticus, its old name is Arthrobacter antarcticus, is a species of bacteria. It is Gram-positive, motile, aerobic and has a rod–coccus cycle. SPC26T (=LMG 24542T =NCCB 100228T) is the type strain.


Illumina dye sequencing

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.
Overview.
Illumina sequencing technology works in three basic steps: amplify, sequence, and analyze. The process begins with purified DNA. The DNA is fragmented and adapters are added that contain segments that act as reference points during amplification, sequencing, and analysis. The modified DNA is loaded onto a flow cell where amplification and sequencing will take place. The flow cell contains nanowells that space out fragments and help with overcrowding. Each nanowell contains oligonucleotides that provide an anchoring point for the adapters to attach. Once the fragments have attached, a phase called cluster generation begins. This step makes about a thousand copies of each fragment of DNA and is done by bridge amplification PCR. Next, primers and modified nucleotides are washed onto the chip. These nucleotides have a reversible 3' fluorescent blocker so the DNA polymerase can only add one nucleotide at a time onto the DNA fragment. After each round of synthesis, a camera takes a picture of the chip. A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip. After each round, non-incorporated molecules are washed away. A chemical deblocking step is then used to remove the 3’ fluorescent terminal blocking group. The process continues until the full DNA molecule is sequenced. With this technology, thousands of places throughout the genome are sequenced at once via massive parallel sequencing.
Procedure.
Genomic Library.
After the DNA is purified a DNA library, genomic library, needs to be generated. There are two ways a genomic library can be created, sonification and tagmentation. With tagmentation, transposases randomly cuts the DNA into sizes between 50 to 500 bp fragments and adds adaptors simultaneously. A genetic library can also be generated by using sonification to fragment genomic DNA. Sonification fragments DNA into similar sizes using ultrasonic sound waves. Right and left adapters will need to be attached by T7 DNA Polymerase and T4 DNA ligase after sonification. Strands that fail to have adapters ligated are washed away.
Adapters.
Adapters contain three different segments: the sequence complementary to solid support (oligonucleotides on flow cell), the barcode sequence (indices), and the binding site for the sequencing primer. Indices are usually six base pairs long and are used during DNA sequence analysis to identify samples. Indices allow for up to 96 different samples to be run together, this is also known as multiplexing. During analysis, the computer will group all reads with the same index together. Illumina uses a "sequence by synthesis" approach. This process takes place inside of an acrylamide-coated glass flow cell. The flow cell has oligonucleotides (short nucleotide sequences) coating the bottom of the cell, and they serve as the solid support to hold the DNA strands in place during sequencing. As the fragmented DNA is washed over the flow cell, the appropriate adapter attaches to the complementary solid support.
Bridge amplification.
Once attached, cluster generation can begin. The goal is to create hundreds of identical strands of DNA. Some will be the forward strand; the rest, the reverse. This is why right and left adapters are used. Clusters are generated through bridge amplification. DNA polymerase moves along a strand of DNA, creating its complementary strand. The original strand is washed away, leaving only the reverse strand. At the top of the reverse strand there is an adapter sequence. The DNA strand bends and attaches to the oligo that is complementary to the top adapter sequence. Polymerases attach to the reverse strand, and its complementary strand (which is identical to the original) is made. The now double stranded DNA is denatured so that each strand can separately attach to an oligonucleotide sequence anchored to the flow cell. One will be the reverse strand; the other, the forward. This process is called bridge amplification, and it happens for thousands of clusters all over the flow cell at once.
Clonal amplification.
Over and over again, DNA strands will bend and attach to the solid support. DNA polymerase will synthesize a new strand to create a double stranded segment, and that will be denatured so that all of the DNA strands in one area are from a single source (clonal amplification). Clonal amplification is important for quality control purposes. If a strand is found to have an odd sequence, then scientists can check the reverse strand to make sure that it has the complement of the same oddity. The forward and reverse strands act as checks to guard against artefacts. Because Illumina sequencing uses DNA polymerase, base substitution errors have been observed, especially at the 3' end. Paired end reads combined with cluster generation can confirm an error took place. The reverse and forward strands should be complementary to each other, all reverse reads should match each other, and all forward reads should match each other. If a read is not similar enough to its counterparts (with which it should be a clone), an error may have occurred. A minimum threshold of 97% similarity has been used in some labs' analyses.
Sequence by synthesis.
At the end of clonal amplification, all of the reverse strands are washed off the flow cell, leaving only forward strands. A primer attaches to the forward strands adapter primer binding site, and a polymerase adds a fluorescently tagged dNTP to the DNA strand. Only one base is able to be added per round due to the fluorophore acting as a blocking group; however, the blocking group is reversible. Using the four-color chemistry, each of the four bases has a unique emission, and after each round, the machine records which base was added. Once the color is recorded the fluorophore is washed away and another dNTP is washed over the flow cell and the process is repeated. dATPs, dTTPs, dGTPs, and dCTPs are washed over the cell separately so each nucleotide is able to be identified.
Starting with the launch of the NextSeq and later the MiniSeq, Illumina introduced a new two-color sequencing chemistry. Nucleotides are distinguished by either one of two colors (red or green), no color ("black") or combining both colors (appearing orange as a mixture between red and green).
Once the DNA strand has been read, the strand that was just added is washed away. Then, the index 1 primer attaches, polymerizes the index 1 sequence, and is washed away. The strand forms a bridge again, and the 3' end of the DNA strand attaches to an oligo on the flow cell. The index 2 primer attaches, polymerizes the sequence, and is washed away.
A polymerase sequences the complementary strand on top of the arched strand. They separate, and the 3' end of each strand is blocked. The forward strand is washed away, and the process of sequence by synthesis repeats for the reverse strand.
Data analysis.
The sequencing occurs for millions of clusters at once, and each cluster has ~1,000 identical copies of a DNA insert. The sequence data is analyzed by finding fragments with overlapping areas, called contigs, and lining them up. If a reference sequence is known, the contigs are then compared to it for variant identification.
This piecemeal process allows scientists to see the complete sequence even though an unfragmented sequence was never run; however, because Illumina read lengths are not very long (HiSeq sequencing can produce read lengths around 90 bp long), it can be a struggle to resolve short tandem repeat areas. Also, if the sequence is de novo and a reference doesn't exist, repeated areas can cause a lot of difficulty in sequence assembly. Additional difficulties include base substitutions (especially at the 3' end of reads) by inaccurate polymerases, chimeric sequences, and PCR-bias, all of which can contribute to generating an incorrect sequence.
Comparison with other sequencing methods.
This technique offers several advantages over traditional sequencing methods such as Sanger sequencing. Sanger sequencing requires two reactions, one for the forward primer and another for the reverse primer. Unlike Illumina, Sanger sequencing uses fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) to determine the sequence of the DNA fragment. ddNTPs are missing the 3' OH group and terminates DNA synthesis permanently. In each reaction tube, dNTPs and ddNTPs are added, along with DNA polymerase and primers. The ratio of ddNTPs to dNTPs matter since the template DNA needs to be completely synthesized, and an overabundance of ddNTPs will create multiple fragments of the same size and position of the DNA template. When the DNA polymerase adds a ddNTP the fragment is terminated and a new fragment is synthesized. Each fragment synthesized is one nucleotide longer than the last. Once the DNA template has been completely synthesized, the fragments are separated by capillary electrophoresis. At the bottom of the capillary tube a laser excites the fluorescently labeled ddNTPs and a camera captures the color emitted.
Due to the automated nature of Illumina dye sequencing it is possible to sequence multiple strands at once and gain actual sequencing data quickly. With Sanger sequencing, only one strand is able to be sequenced at a time and is relatively slow. Illumina only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques (i.e. pyrosequencing).
Examples of use.
Illumina sequencing has been used to research transcriptomes of the sweet potato and the gymnosperm genus "Taxus".


Synchronous culture

A synchronous or synchronized culture is a microbiological culture or a cell culture that contains cells that are all in the same growth stage.
All cells in the synchronously growing culture divide at a time, they yield a Zig Zag pattern of growth curve.
As numerous factors influence the cell cycle (some of them stochastic) normal cultures have cells in all stages of the cell cycle. Obtaining a culture with a unified cell-cycle stage is useful for biological research where a particular stage in the cell cycle is desired (such as the culturing of parasitized cells). Since cells are too small for certain research techniques, a synchronous culture can be treated as a single cell; the number of cells in the culture can be easily estimated, and quantitative experimental results can simply be divided in the number of cells to obtain values that apply to a single cell. Synchronous cultures have been extensively used to address questions regarding cell cycle and growth, and the effects of various factors on these.
Methods.
Synchronous cultures can be obtained in several ways:


Olga Dontsova

Olga Anatolyevna Dontsova (born January 7, 1959) is a Russian biochemist and an academician at the Russian Academy of Sciences. Her research interests includes: structure and functions of RNA-containing cellular machines, functional properties and mechanisms of regulation of Telomerase Ribonucleoprotein particle (RNP) complexes and non-coding RNAs.
Education and work.
In 1991 she defended her Ph.D. from the Chemistry Department of Moscow State University and in 1997 her doctoral dissertation "development of chemical methods for studying the structure and function of complex ribonucleoprotein systems" was published. Since 1999 she is the professor of Bio-organic chemistry, Department of Chemistry of Natural Compounds at Faculty of Chemistry, Moscow State University.
She is the member of the Russian Foundation for Basic Research Board.


Abgent

Abgent is a global biotechnology company based in San Diego, California, US with offices in Maidenhead, UK and Suzhou, China and distributors around the world. Abgent develops antibodies and related agents to study proteins involved in cellular function and disease. Abgent's antibodies target key areas of research including autophagy, neuroscience, cancer, stem cells and more. Abgent was acquired in 2011 by WuXi AppTec, a global pharmaceutical, biopharmaceutical, and medical device outsourcing company with operations in China and the United States.
Peer review.
Abgent was listed as a selected supplier in "Nature Magazine", "Antibody Technology", "Drug Discovery Features" and "The Scientist's" cell signaling feature. More than 1,100 peer-reviewed publications in scientific journals have cited Abgent antibody, protein, and peptide products and custom services.
Core business.
As one of the world's largest manufacturers of antibodies for biological research and drug discovery, Abgent develops, produces, and sells antibodies for use in academic, biotechnological, and pharmaceutical industries. Core products are complemented by custom antibody services and custom protein services for drug discovery targets.
Tools.
SUMOplot Analysis Program.
"SUMOplot" is a tool used to predict sumoylation sites, an important post-translational modification of proteins. SUMO-modified proteins contain the tetrapeptide motif B-K-x-D/E where B is a hydrophobic residue, K is the lysine conjugated to SUMO, x is any amino acid (aa), D or E is an acidic residue. Substrate specificity appears to be derived directly from Ubc9 and the respective substrate motif. SUMOplot predicts the probability for the SUMO consensus sequence (SUMO-CS) to be engaged in SUMO attachment. The SUMOplot score system is based on two criteria: first, direct amino acid match to the SUMO-CS observed and shown to bind Ubc9, and second, substitution of the consensus amino acid residues with amino acid residues exhibiting similar hydrophobicity. SUMOplot has been used in the past to predict Ubc9 dependent sites.
Autophagy Receptor Motif Plotter.
The autophagy pathway is mediated by selective receptors. They recognize and sort diverse cargo substrates (e.g., proteins, organelles, pathogens) for delivery to the autophagic machinery. Known autophagy receptors are characterized by short linear sequence motifs (autophagy receptor motifs, or ARMs) responsible for the interaction with the Atg8/LC3 family. Many ARM-containing proteins (ARM-CPs) are also involved in autophagosome formation and maturation and a few of them in regulating signaling pathways. "Autophagy Receptor Motif Plotter" assists in the identification of novel ARM-CPs. Users input a given an amino acid sequence into the web-enabled tool, and the program identifies internal sequences matching a pattern within the 3 classes of the extended ARM motif (x6-W/F/Yxxx-x2). The program then computes and lists the top four scores for each motif class (W-, F-, Y-). The full sequence of the ARM-CP is displayed, where ARMs are colored by their score and ranked score-values are presented in tabular form.


Osmoprotectant

Osmoprotectants or compatible solutes are small organic molecules with neutral charge and low toxicity at high concentrations that act as osmolytes and help organisms survive extreme osmotic stress. Osmoprotectants can be placed in three chemical classes: betaines and associated molecules, sugars and polyols, and amino acids. These molecules accumulate in cells and balance the osmotic difference between the cell's surroundings and the cytosol. In plants, their accumulation can increase survival during stresses such as drought. In extreme cases, such as in bdelloid rotifers, tardigrades, brine shrimp, and nematodes, these molecules can allow cells to survive being completely dried out and let them enter a state of suspended animation called cryptobiosis.
Intracellular osmoprotectant concentrations are regulated in response to environmental conditions such as osmolarity and temperature via regulation of specific transcription factors and transporters. They have been shown to play a protective role by maintaining enzyme activity through freeze-thaw cycles and at higher temperatures. It is currently believed that they function by stabilizing protein structures by promoting preferential exclusion from the water layers on the surface of hydrated proteins. This favors the native conformation and displaces inorganic salts that would otherwise cause misfolding.
Role.
Compatible solutes have a functional role in agriculture. In high stress conditions, such as drought or high salinity, plants that naturally create or take up osmoprotectants show increased survival rates. By inducing expression or uptake of these molecules in crops in which they are naturally not present, there is an increase in the areas in which they are able to be grown. One documented reason for increased growth is regulation of toxic reactive oxygen species (ROS). In high salinity ROS production is stimulated by the photosystems of the plant. Osmoprotectants can prevent the photosystem-salt interactions, reducing ROS production. For these reasons, introduction of biosynthetic pathways which result in the creation of osmoprotectants in crops is a current area of research, but inducing expression at significant amounts is currently posing a barrier in this area of research.
Osmoprotectants are also important for the maintenance of top soil bacteria populations. Desiccation of top soils results in increased salinity. In these situations, the soil microbes increase the concentration of these molecule in their cytoplasm into the molar range allowing them to persist until conditions approve. In extreme cases, osmoprotectants allow cells to enter cryptobiosis. In this state the cytosol and osmoprotectants become a glass-like solid that helps stabilize proteins and cell membranes from the damaging effects of desiccation.
Additionally, osmoprotectants provide a method to regulate gene expression in response to environmental osmolarity. The presence of compatible solutes even in small concentrations has been shown to affect gene expression. Their effects range from inducing production of more compatible solutes to regulating components involved in infection, such as phospholipase C in "Pseudomonas aeruginosa".


Elizabeth Nolan

Elizabeth Marie Nolan (born 1978) is an American chemist and Associate Professor at Massachusetts Institute of Technology.
Early life and education.
Nolan was born in Niskayuna, New York. She studied at Smith College, where she majored in chemistry and graduated magna cum laude in 2000. During her undergraduate studies she minored in music and worked with Robert Linck on computational chemistry. She studied the stereoelectronic effects in substituted alkanes. Nolan was a member of Phi Beta Kappa. She was awarded a Fulbright Program Scholarship and moved to France to study siderophore-iron complexes. Nolan moved to Massachusetts Institute of Technology for her graduate studies, where she was supervised by Stephen J. Lippard. She developed small molecule fluorescent sensors to monitor for zinc in neurobiology and mercury in aqueous solutions. Together they filed a patent for Fluorescein-based metal sensors. Nolan was a postdoctoral scientist at the Harvard Medical School, working with Christopher T. Walsh on the biosynthetic assembly of microcin E492m. Microcin E492m is an antibiotic peptide that can target Gram-negative bacteria which express siderophore transporters. She was awarded a $2.5 million National Institutes of Health grant in 2010 to study antibacterial peptides and zinc in innate immunity. In 2011 she contributed to the book "Letters to a Young Chemist."
Research and career.
Nolan was appointed as an Assistant Professor at the Department of Chemistry at the Massachusetts Institute of Technology in 2014. She explores the coordination chemistry of metal ions in biological systems; in particular how proteins destroy microbes by denying them metal nutrients. She looks at the peptides and metalloproteins that are involved in mammalian immune response. Her current focus is on calprotectins and how they bind metals. She used magnetic circular dichroism to study the binding of iron to human calprotectin. Her group look to understand how Neuronal cells process and removes SOD1 point mutants.
Nolan looks to design drugs to fight bacterial infections. They study how immunity peptides that are encoded by the gene clusters that biosynthesize antibiotics which use metal ion transporters protect the organisms that produce them. She proposes that hijacking the siderophore uptake pathways could allow new prevention and treatment against diseases. She worked with Manuela Raffatellu at University of California, Irvine to develop a new immunisation strategy against salmonella. They target siderophores, a molecule that salmonella secretes to scavenge iron. Immunisation against siderophores led to the production of antibodies that reduced the growth of salmonella and other bacteria. She is on the editorial board of "Cell Chemical Biology".


Sergei Popov (bioweaponeer)

Sergei Popov is a scientist and bioweaponeer formerly in the Soviet biological weapons program.
He defected to the West in 1992 and now lives and works in the United States.
Biography.
After obtaining his degree in biochemistry, Popov served as a division head at the State Research Center of Virology and Biotechnology (known as "Vector") and at Obolensk, both branches of the Soviet bio-weapons program dedicated to developing genetically enhanced products. His position led him to expand his researches into the fields of molecular biology and microbiology.
Popov worked at Vector from 1976 to 1986 and at Obolensk from 1986 until 1992. His work included "designer" bio-agents that would cause the symptoms of lupus and rheumatoid arthritis, in which a victim's auto-immune system attacks its own body. His team inserted genes into viruses to make protein fragments of myelin (the sheathing around nerves). Victims that became infected would develop multiple sclerosis, a degenerative disease of the nervous system. By splicing myelin into "Legionella" (Legionnaires' disease), they also created an agent that caused brain damage, paralysis and death. The recombinant "Legionella" was very infectious and lethal with only a few cells causing disease.
At Oblensk, Popov and his team spliced the diphtheria toxin gene into the plague bacterium, thus creating a highly virulent and deadly strain.
Popov has described Biopreparat's "Project Bonfire", whose goal was to develop antibiotic-resistant microbial strains, and "Project Factor", whose goal was to create microbial weapons with new biologic properties that would result in high virulence, improved stability, and new clinical syndromes.
In 1992, Popov defected to the United Kingdom and later traveled to the United States. He worked for Hadron, Inc. in microbiology and pharmacology and at George Mason University.


Manju Bansal

Manju Bansal (born. 1 December 1950) specializes in the field of Molecular biophysics and is currently a professor of theoretical Biophysics group in Molecular Biophysics unit of in the Indian Institute of Science, Bangalore. She is the founder director of the Institute of Bioinformatics and Applied Biotechnology at Bangalore.
Education.
Bansal did her schooling from Hyderabad and Dehradun. She developed a great interest in Science and later went ahead to earn her BSc and MSc from Osmania University, Hyderabad.
In 1972 she joined the Molecular Biophysics Unit, Indian Institute of Science, Bangalore for her doctoral degree. She got an opportunity to work under the guidance of biophysicist G. N. Ramachandran, on the theoretical modeling of the triple helical structure of the fibrous protein collagen. She received her PhD in 1977.
Thereafter, she continued working at IISc as a post-doctoral fellow on left handed and other unusual structures of DNA till 1981. She then went to Germany as an Alexander von Humboldt Fellow at the European Molecular Biology Laboratory, Heidelberg, for a year and worked on the structure of filamentous phages.
Fellowships.
Bansal has been awarded an EMBL Visiting Fellowship and AvH Fellowship, Germany and Senior Fulbright Fellowship, USA. She has been a Visiting Professor at Rutgers University, USA, and Visiting Consultant at NIH, Bethesda, USA.
Awards & Honours.
Bansal was awarded the INSA Medal for Young Scientists in 1979.
she is a Fellow of the Indian Academy of Sciences, Bangalore and National Academy of Sciences (India), Allahabad, since 1998.


5-Ethyl-DMT

5-Ethyl-"N","N"-dimethyltryptamine is a tryptamine derivative which acts as an agonist at the 5-HT1A and 5-HT1D serotonin receptors, with around 3x selectivity for 5-HT1D.


BTBD9

BTBD9 is a human gene.
BTBD9 encodes a protein that is in a subgroup of BTB(POZ) proteins, which contribute to the forming of limbs and determination of cell fate in developing Drosophila melanogaster. BTB(POZ) proteins also play a role in cellular functions such as: cytoskeleton regulation, transcription regulation, the gating and assembly of ion channels, and ubiquitination of proteins. BTBD9 is highly expressed throughout the brain and shows variable levels of expression in most other body tissues.
The gene is located on the short arm of chromosome 6. This domain is understood to contain genes encoding protein-protein interactions.
Research Models.
There are extensive homologs to BTBD9 which allow for the use of animal models in deciphering its functions and interactions. The BTBD9 homolog Btbd9 is extensively expressed in the central nervous system of adult mice including the thalamus, sub-thalamic nuclei, cerebral cortex, cerebellum, hippocampus, and caudate nucleus. The Drosophila homolog dBTBD9, was shown to regulate dopamine levels in the Drosophila brain and iron regulation in human cell-lines.
Synaptic Plasticity.
A recent study using Btbd9 knockout mice argued that BTBD9 is involved in synaptic plasticity, learning and memory, and protein alterations associated with vesicle recycling and endocytosis.
Clinical Relevance.
There is some evidence that BTBD9 may be associated with Restless legs syndrome. And mutations to BTBD9 are positively correlated with characteristic symptoms of Restless leg syndrome such as decreased dopamine levels, increased movement, and disrupted sleep patterns. Some genetic variants in BTBD9 that have been linked to Restless leg syndrome are also correlated with Tourette’s Syndrome that doesn’t present with Obsessive Compulsive Disorder.


Ubiquitin bacterial

Ubiquitin Bacterial (UBact) is a protein that is homologous to Prokaryotic ubiquitin-like protein (Pup). UBact was recently described by the group of Professor Aaron Ciechanover at the Technion, Israel.
Ubiquitin was named for its ubiquitous presence among eukaryotes, while UBact ('Ubiquitin bacterial') is very limited in occurrence among the vast number of bacterial species. The terms 'Ubiquitin Bacterial' and 'Prokaryotic ubiquitin-like protein' suggest a molecular similarity between ubiquitin and UBact/Pup which is largely absent. While ubiquitin assumes a highly stable three-dimensional structure in solution, Pup has been shown to belong to the group of intrinsically disordered proteins.
The establishment of the term UBact is controversial, since to date there is no experimental evidence presented to justify the distinction of UBact from Pup. The term UBact was denoted because several bacterial species from the phylum Nitrospirae (where UBact was initially identified; e.g., "Leptospirillum ferriphilum") contain both the Pup-proteasome system and a novel ORF-proteasome system that needed to be addressed and therefore was denoted UBact. The conjugation-proteasome components neighboring the UBact and Pup loci in these Nitrospirae bacteria show weak similarity and are probably not entirely redundant. Figure 2 illustrates the differences between the "UBact" and "Pup" loci in the representative Nitrospirae bacterium "Leptospirillum ferrodiazotrophum". Further analyses of the UBact (and not Pup) locus in "Leptospirillum ferrodiazotrophum" revealed its existence and extreme conservation across several gram-negative bacterial phyla, as illustrated in figure 3.
In spite of the large difference in sequence, UBact is homologous to Pup and shares several characteristics with it: (i) same genomic location within a cluster of genes homologous to "Mpa" -> "Dop" -> "Pup/UBact" -> "PrcB" -> "PrcA" -> "PafA", (ii) C-terminal sequence that ends exclusively with glutamine or glutamate across bacterial species, (iii) short size (similar to that of ubiquitin) and, (iv) high sequence conservation across tremendous evolutionary distance (a characteristic also in common with ubiquitin). The differences between UBact and Pup are their taxonomic distribution and amino acid sequences. While Pup is predominantly found in the gram-positive phylum Actinobacteria, UBact was identified only in gram-negative bacteria from the following five phyla: Nitrospirae, Verrucomicrobia, Armatimonadetes, Nitrospinae, and Planctomycetes. UBact was also identified in the genomes of several candidatus bacteria, and specifically from the candidate divisions Acetothermia, Handelsmanbacteria, Fraserbacteria, Terrybacteria, Poribacteria, Parcubacteria and Yanofskybacteria. With regard to the amino acid sequence, in difference from Pup and Ubiquitin, UBact does not contain a di-glycine motif at its C-terminus. Rather, it usually ends with the sequence R[T/S]G[E/Q] (see figure 3).
It took almost ten years since the discovery of Pup in 2008, to identify UBact. This is probably due to the difference between Pup and UBact amino acids sequences, and because very few bacteria from the five phyla where UBact is found have been sequenced.
Bacteria from the phyla where UBact is found interact with humans, and are found in the human gut microbiota. In marine systems, the most frequently encountered nitrogen-oxidizing bacteria are related to the UBact encoding "Nitrospina gracilis" From the knowledge accumulated about the Pup-proteasome system and its importance in bacterial durability and disease causing ability, the homologous UBact-proteasome system is expected to have similar impact on the gram-negative bacteria where it is found. In addition to humans, animals such livestock and fish that eat from the ground or swim in water are expected to be constantly exposed to UBact containing bacteria in the soil and water respectively.
From evolutionary perspective, the finding of the UBact-proteasome system in gram-negative bacteria suggests that either the Pup/UBact-proteasome systems evolved in bacteria prior to the split into gram positive and negative clades over 3000 million years ago or, that these systems were acquired by different bacterial lineages through horizontal gene transfer(s) from a third, yet unknown, organism. In support of the second possibility, two UBact loci were found in the genome of an uncultured anaerobic methanotrophic Archaeon (ANME-1;locus CBH38808.1 and locus CBH39258.1). More possibilities exist.
Update: UBact is also found in the gram-negative bacterial phylum Gemmatimonadetes (e.g., A0A2E8WA32, A0A2E3J6F7, A0A2E7JSE3) in the candidate phylum Latescibacteria (previously known as WS3; e.g., A0A3D2RHP4, A0A3D5FTR6, A0A3D4H075, and A0A3B8MMW3) and in the phylum Abditibacteriota (previously candidate phylum FBP; e.g., A0A2S8SU03).


Dystrophin-associated protein

A dystrophin-associated protein is a protein that helps to form the connection between intracellular dystrophin and the extracellular basal lamina.
Examples include sarcoglycan and dystroglycan.


Anti-Q RNA

Anti-Q RNA (formerly Qa RNA) is a small ncRNA from the conjugal plasmid pCF10 of "Enterococcus faecalis". It is coded in cis to its regulatory target, prgQ, but can also act in trans. Anti-Q is known to interact with nascent prgQ transcripts to allow formation of an intrinsic terminator, or attenuator, thus preventing transcription of downstream genes. This mode of regulation is essentially the same as that of the countertranscript-driven attenuators that control copy number in pT181, pAMbeta1 and pIP501 and related Staphylococcal plasmids.
Anti-Q is transcribed from the same segment of DNA as prgQ, except from the opposite strand, making it perfectly complementary to a portion of prgQ. Further experiments have experimentally confirmed the original consensus secondary structure and demonstrated that only certain regions of Anti-Q interact with prgQ.
Anti-Q is derived from the 5’ end a longer transcript. The 3’ end of this transcript encodes PrgX, a repressor of prgQ transcription.


Isolecithal

Isolecithal ("Greek" iso = equal, lekithos = yolk) refers to the even distribution of yolk in the cytoplasm of ova of mammals and other vertebrates, notably fishes of the families Petromyzontidae, Amiidae, and Lepisosteidae. Isolecithal cells have two equal hemispheres of yolk. However, during cellular development, normally under the influence of gravity, some of the yolk settles to the bottom of the egg, producing an uneven distribution of yolky hemispheres. Such uneven cells are known as telolecithal and are common where there is sufficient yolk mass.
In the absence of a large concentration of yolk, four major cleavage types can be observed in isolecithal cells: radial holoblastic, spiral holoblastic, bilateral holoblastic, and rotational holoblastic cleavage. These holoblastic cleavage planes pass all the way through isolecithal zygotes during the process of cytokinesis. Coeloblastula is the next stage of development for eggs that undergo this radial cleavage. In mammals, because the isolecithal cells have only a small amount of yolk, they require immediate implantation onto the uterine wall to receive nutrients.


DAOY (biology)

DAOY is a widely used human primary medulloblastoma cell line. It has epithelial morphology and was obtained from a 4-year-old Caucasian boy in 1985. It can be obtained from the American Type Culture Collection (ATCC) where it is known by the catalog number HTB-186. Its proteome and phenotype have been investigated in many publications.
DAOY is most commonly grown in Eagle's minimal essential medium with 10% FBS and antibiotics. It is incubated at 37 degrees Celsius in a 5% carbon dioxide atmosphere.


MMP26

Matrix metalloproteinase-26 also known as matrilysin-2 and endometase is an enzyme that in humans is encoded by the "MMP26" gene.
Function.
Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. The encoded protein degrades type IV collagen, fibronectin, fibrinogen, casein, vitronectin, alpha 1-antitrypsin (A1AT), alpha 2-macroglobulin (A2M), and insulin-like growth factor-binding protein 1 (IGFBP), and activates MMP9 by cleavage. The protein differs from most MMP family members in that it lacks a conserved C-terminal protein domain.


Clara Benson

Clara Cynthia Benson (1875–1964) was a Canadian chemist, the sole female founder of the American Society for Biological Chemistry (now the American Society for Biochemistry and Molecular Biology (ASBMB)) and one of the first two women to earn a Ph.D. from the University of Toronto (U of T) (the other being philosophy student Emma Baker). She later became one of U of T's first two female associate professors. Trained in physical chemistry, she switched focus to biochemistry when lack of job opportunities for female chemists led her to take a position teaching food chemistry as part of U of T's Domestic Science program. She also played a large role in the development of U of T's women's athletics program.
Early life and education.
Clara Cynthia Benson was born in Port Hope, Ontario, Canada June 5, 1875 to Thomas Moore Benson and Laura Abigail Fuller. Laura and Thomas, a widowed businessman, lawyer, and judge, had three children together and additionally raised two daughters from Thomas' first marriage. Clara attended Port Hope High School then entered University College of the University of Toronto (U of T) in 1895 to study chemistry, mathematics and physics. This was only one year after the school began admitting women, and women were still not allowed into the school's reading rooms and were denied access to the library catalogues.
Benson graduated with a Bachelor of Arts in chemistry from U of T in 1899 (the first woman to do so) and continued straight into PhD studies. She earned her doctorate in 1903, making her one of the first two women to earn a Ph.D. from the University of Toronto (U of T) (the other being philosophy student Emma Baker).
Benson's doctoral research, supervised by William Lash Miller, examined reaction rates of inorganic salt solutions. Her thesis, "The rates of the reactions in solutions containing ferrous sulphate, potassium iodide, and chromic acid" was published in the "Journal of Physical Chemistry" ("JPC") in May, 1903.
Career and research.
Benson's early research was in the field of physical chemistry, with an emphasis on reaction rates of inorganic salt solutions. Her 1902 article "The Rate of Oxidation of Ferrous Salts by Chromic Acid" may make her the second female author (after Marie Curie) to publish in the "Journal of Physical Chemistry" ("JPC").
As a woman, she had difficulty finding a job as a physical chemist after graduating, so she took a position as a demonstrator in food science at U of T's new Lillian Massey School of Domestic Science. She objected to this program's goal of preparing women to be housewives and had even signed a petition organized by University College's Women's Alumnae Association in 1902 questioning the program's introduction. At the time, however, food chemistry was one of the chemistry sub-fields with better professional opportunities for women.
This position involved switching from U of T's Chemistry Department to their Physiology (Physiological Chemistry) Department, where she was mentored by the "Father of the Medical School at Toronto," A.B. Macallum. Her subsequent research included biochemical examinations of fluid and tissue composition.
When food science was incorporated into U of T's medical curriculum in 1905, Benson was promoted to lecturer in physiological chemistry (biochemistry), making her the first woman at U of T to achieve a rank above demonstrator. In 1906, a royal commission report led to the creation of the Faculty of Household Science, of which Benson and the principal, Annie Laird, became associate professors, making them U of T's first female professors. Benson helped develop the school's food chemistry program and, in 1926, was promoted to full professor and head of the Department of Food Chemistry (a position she held until her retirement as professor emeritus in 1945).
Starting in 1915, she conducted summer studies at St. Andrews Biological Station examining the chemistry of seafood. At the request of Canada's Ministry of Marine and Fisheries, which was trying to build consumer demand for fish, she organized a group of food scientists from Canadian universities to work to improve fish preparation methods.
During World War I she developed and organized a course of instruction on ways to adapt food chemistry analysis techniques to explosives. These methods were adopted by munitions laboratories, helping standardized their production steps.
She was sole female founder of the American Society for Biological Chemistry (now the American Society for Biochemistry and Molecular Biology (ASBMB)) when it formed in December, 1906.
Other interests and later life.
Benson advocated for the development of women's athletics at the University of Toronto, co-chairing a committee on the matter and serving as the first president of the Women's Athletic Association from 1921 until her retirement. She sat on a committee of female faculty members formed in 1928 to fight for the creation of a women's athletic facility. When U of T opened their first women's gymnasium in 1959, they named it the Benson Building in her honor.
Benson served on the national board of the YWCA (Young Women's Christian Association), chairing their Foreign Committee, and her work with the organization led her to sponsor two French World War II orphans after her retirement. Her hobbies included stamp-collecting and traveling. She also enjoyed film-making, and videos she took while on some of her travels are housed at U of T's archives.
She was colleagues and friends with biochemist Maud Menten, who was also trained by Archibald Macallum.
Benson never married nor had children, and after retiring in 1945, she returned to Port Hope where she died March 24, 1964 (aged 89).
Honors and awards.
Benson was elected a fellow of the Canadian Institute of Chemistry in 1919, but was not allowed to attend their Annual Dinner in 1920 because she was a woman.
She was listed in the 1920s' "American Men in Science".
U of T's Household Science alumnae created a fellowship in her honor in 1950 and hung a portrait of her by Yousef Karsh in the Household Science building.
In 1992, the Canadian Society of Chemistry created the annually-awarded Clara Benson Award to honor female chemists working in Canada.
In 2003, U of T celebrated the 100 year anniversary of her PhD achievement with a day of celebrations including a reenactment of her thesis defense.


MMP27

Matrix metallopeptidase 27 also known as MMP-27 is an enzyme which in humans is encoded by the "MMP27" gene.
Structure.
MMP-27 was discovered and cloned in 1998 by Yang and Kurkinen. Initially compared to the so-called Chicken MMP (CMMP), MMP-27 actually shows very little sequence homology with this protease. Sequence homology predicts that the human MMP-27 gene encodes the canonical domains shared by most MMPs (annotation based on Uniprot entry Q9H306): (i) a signal peptide (residues 1-17), (ii) a propeptide (18-98) containing the cysteine switch motif (89-96), (iii) a catalytic domain (99-263) containing the typical HEXXHXXGXXH motif of the metzincins (M10 and M12 families of the MEROPS[2] database), (iv) a proline-rich hinge region (264-278) and (v) a hemopexin-like domain (279-465) folded as a four-bladed β-propeller through disulfide bond formation between the two flanking Cys residues (Cys279 and Cys465). MMP-27 could be classified in the stromelysin group of MMPs, since MMP-27 shows 51,6% homology with stromelysin-2 (MMP-10) and localizes in the cluster of MMPs located on chromosome 11.
Like the six known MT-MMPs, human MMP-27 is prolonged by an additional C-terminal domain (466-513). The Spoctopus algorithm for topological prediction suggests that this C-terminal extension (CTE) includes a potential transmembrane domain (490-510). However, this sequence is less hydrophobic than in transmembrane MT-MMPs (MMP-14, -15, -16 and -24) as it contains hydrophilic/charged residues, in particular His492, Lys493, His504 and Lys507.
Function.
Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMPs are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases.
Cominelli A. and colleagues demonstrated that MMP-27 is an unusual protease which is not secreted and is efficiently retained in the endoplasmic reticulum in three mammalian cell lines. Deletion mutants and swapping with recombinant MMP-10 demonstrate that the unique MMP-27 C-terminal extension (CTE) is necessary and sufficient for endoplasmic reticulum retention but does not provide a stable membrane anchorage. Despite sequence homology with MT-MMPs, the CTE is not a transmembrane domain and does not interact permanently with membrane. This unique feature for an MMP raises important questions about potential functions of MMP-27, which remains to be investigated.
Clinical significance.
Sparse information about MMP-27 expression was found in studies of gene expression profiling (micro-array) or in expression pattern analysis of MMP family members during developmental, physiological or pathological processes. MMP-27 transcript is detected in almost every tissue, except the brain, with the highest expression found in the liver during mouse development In the adult, MMP-27 mRNA is mostly abundant in anti-IgG/IgM stimulated B lymphocytes, bone and kidney but is present at lower levels in the heart.
A recent investigation of the transcriptome from distinct tissue compartments of the menstrual endometrium disclosed specific MMP-27 overexpression in areas of stromal breakdown. In another transcriptomic study, MMP-27 was found to be increased in the human endometrium at the end of the secretory phase, before menstruation. Moreover, MMP-27 expression is down-regulated in macrophages when co-cultured with ovarian cancer cells but up-regulated in cartilages from patients with osteoarthritis or in abdominal aortic aneurysms. MMP-27 was also identified, at the protein level, in MDA-MB-231 breast cancer cell line and in primary human breast cancer. Recently, MMP-27 has been demonstrated to be expressed by CD163+/CD206+ macrophages in the human endometrium and in superficial endometriotic lesions.