title: MicroRNA

text: The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. Perfect or near perfect base pairing with the target RNA promotes cleavage of the RNA. This is the primary mode of plant miRNAs. In animals the match-ups are imperfect.


title: MicroRNA

text: For partially complementary microRNAs to recognise their targets, nucleotides 2–7 of the miRNA (its 'seed region') must be perfectly complementary. Animal miRNAs inhibit protein translation of the target mRNA (this is present but less common in plants). Partially complementary microRNAs can also speed up deadenylation, causing mRNAs to be degraded sooner. While degradation of miRNA-targeted mRNA is well documented, whether or not translational repression is accomplished through mRNA degradation, translational inhibition, or a combination of the two is hotly debated. Recent work on miR-430 in zebrafish, as well as on bantam-miRNA and miR-9 in "Drosophila" cultured cells, shows that translational repression is caused by the disruption of translation initiation, independent of mRNA deadenylation.


title: MicroRNA

text: miRNAs occasionally also cause histone modification and DNA methylation of promoter sites, which affects the expression of target genes.


title: MicroRNA

text: It is often impossible to discern these mechanisms using experimental data about stationary reaction rates. Nevertheless, they are differentiated in dynamics and have different "kinetic signatures".


title: MicroRNA

text: Unlike plant microRNAs, the animal microRNAs target diverse genes. However, genes involved in functions common to all cells, such as gene expression, have relatively fewer microRNA target sites and seem to be under selection to avoid targeting by microRNAs. There is a strong correlation between "ITPR" gene regulations and mir-92 and mir-19.


title: MicroRNA

text: dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters can induce potent transcriptional activation of associated genes. This was demonstrated in human cells using synthetic dsRNAs termed small activating RNAs (saRNAs), but has also been demonstrated for endogenous microRNA.


title: MicroRNA

text: Interactions between microRNAs and complementary sequences on genes and even pseudogenes that share sequence homology are thought to be a back channel of communication regulating expression levels between paralogous genes (genes having a similar structure indicating divergence from a common ancestral gene). Given the name "competing endogenous RNAs" (ceRNAs), these microRNAs bind to "microRNA response elements" on genes and pseudogenes and may provide another explanation for the persistence of non-coding DNA.


title: MicroRNA

text: Some researches show that mRNA cargo of exosomes may have a role in implantation, they can savage an adhesion between trophoblast and endometrium or support the adhesion by down regulating or up regulating expression of genes involved in adhesion/invasion.


title: MicroRNA

text: miRNAs are well conserved in both plants and animals, and are thought to be a vital and evolutionarily ancient component of gene regulation. While core components of the microRNA pathway are conserved between plants and animals, miRNA repertoires in the two kingdoms appear to have emerged independently with different primary modes of action.


title: MicroRNA

text: microRNAs are useful phylogenetic markers because of their apparently low rate of evolution. microRNAs' origin as a regulatory mechanism developed from previous RNAi machinery that was initially used as a defense against exogenous genetic material such as viruses. Their origin may have permitted the development of morphological innovation, and by making gene expression more specific and 'fine-tunable', permitted the genesis of complex organs and perhaps, ultimately, complex life. Rapid bursts of morphological innovation are generally associated with a high rate of microRNA accumulation.


title: MicroRNA

text: New microRNAs are created in multiple ways. Novel microRNAs can originate from the random formation of hairpins in "non-coding" sections of DNA (i.e. introns or intergene regions), but also by the duplication and modification of existing microRNAs. microRNAs can also form from inverted duplications of protein-coding sequences, which allows for the creation of a foldback hairpin structure. The rate of evolution (i.e. nucleotide substitution) in recently originated microRNAs is comparable to that elsewhere in the non-coding DNA, implying evolution by neutral drift; however, older microRNAs have a much lower rate of change (often less than one substitution per hundred million years), suggesting that once a microRNA gains a function, it undergoes purifying selection. Individual regions within an miRNA gene face different evolutionary pressures, where regions that are vital for processing and function have higher levels of conservation. At this point, a microRNA is rarely lost from an animal's genome, although newer microRNAs (thus presumably non-functional) are frequently lost. In "Arabidopsis thaliana", the net flux of miRNA genes has been predicted to be between 1.2 and 3.3 genes per million years. This makes them a valuable phylogenetic marker, and they are being looked upon as a possible solution to outstanding phylogenetic problems such as the relationships of arthropods. On the other hand, in multiple cases microRNAs correlate poorly with phylogeny, and it is possible that their phylogenetic concordance largely reflects a limited sampling of microRNAs.


title: MicroRNA

text: microRNAs feature in the genomes of most eukaryotic organisms, from the brown algae to the animals. However, the difference in how these microRNAs function and the way they are processed suggests that microRNAs arose independently in plants and animals.


title: MicroRNA

text: Focusing on the animals, the genome of "Mnemiopsis leidyi" appears to lack recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. It is the only animal thus far reported to be missing Drosha. MicroRNAs play a vital role in the regulation of gene expression in all non-ctenophore animals investigated thus far except for "Trichoplax adhaerens", the only known member of the phylum Placozoa.


title: MicroRNA

text: Across all species, in excess of 5000 different miRNAs had been identified by March 2010. Whilst short RNA sequences (50 – hundreds of base pairs) of a broadly comparable function occur in bacteria, bacteria lack true microRNAs.


title: MicroRNA

text: While researchers focused on miRNA expression in physiological and pathological processes, various technical variables related to microRNA isolation emerged. The stability of stored miRNA samples has been questioned. microRNAs degrade much more easily than mRNAs, partly due to their length, but also because of ubiquitously present RNases. This makes it necessary to cool samples on ice and use RNase-free equipment.


title: MicroRNA

text: microRNA expression can be quantified in a two-step polymerase chain reaction process of modified RT-PCR followed by quantitative PCR. Variations of this method achieve absolute or relative quantification. miRNAs can also be hybridized to microarrays, slides or chips with probes to hundreds or thousands of miRNA targets, so that relative levels of miRNAs can be determined in different samples. microRNAs can be both discovered and profiled by high-throughput sequencing methods (microRNA sequencing). The activity of an miRNA can be experimentally inhibited using a locked nucleic acid (LNA) oligo, a Morpholino oligo or a 2'-O-methyl RNA oligo. A specific miRNA can be silenced by a complementary antagomir. microRNA maturation can be inhibited at several points by steric-blocking oligos. The miRNA target site of an mRNA transcript can also be blocked by a steric-blocking oligo. For the "in situ" detection of miRNA, LNA or Morpholino probes can be used. The locked conformation of LNA results in enhanced hybridization properties and increases sensitivity and selectivity, making it ideal for detection of short miRNA.


title: MicroRNA

text: High-throughput quantification of miRNAs is error prone, for the larger variance (compared to mRNAs) that comes with methodological problems. mRNA-expression is therefore often analyzed to check for miRNA-effects in their levels (e.g. in). Databases can be used to pair mRNA- and miRNA-data that predict miRNA-targets based on their base sequence. While this is usually done after miRNAs of interest have been detected (e. g. because of high expression levels), ideas for analysis tools that integrate mRNA- and miRNA-expression information have been proposed.


title: MicroRNA

text: Just as miRNA is involved in the normal functioning of eukaryotic cells, so has dysregulation of miRNA been associated with disease. A manually curated, publicly available database, miR2Disease, documents known relationships between miRNA dysregulation and human disease.


title: MicroRNA

text: The first human disease known to be associated with miRNA deregulation was chronic lymphocytic leukemia. Many other miRNAs also have links with cancer and accordingly are sometimes referred to as "oncomirs". In malignant B cells miRNAs participate in pathways fundamental to B cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches and the production and class-switching of immunoglobulins. MiRNAs influence B cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells.


title: MicroRNA

text: Another role for miRNA in cancers is to use their expression level for prognosis. In NSCLC samples, low miR-324a levels may serve as an indicator of poor survival. Either high miR-185 or low miR-133b levels may correlate with metastasis and poor survival in colorectal cancer.


title: MicroRNA

text: Furthermore, specific miRNAs may be associated with certain histological subtypes of colorectal cancer. For instance, expression levels of miR-205 and miR-373 have been shown to be increased in mucinous colorectal cancers and mucin-producing Ulcerative Colitis-associated colon cancers, but not in sporadic colonic adenocarcinoma that lack mucinous components. In-vitro studies suggested that miR-205 and miR-373 may functionally induce different features of mucinous-associated neoplastic progression in intestinal epithelial cells.


title: MicroRNA

text: Hepatocellular carcinoma cell proliferation may arise from miR-21 interaction with MAP2K3, a tumor repressor gene. Optimal treatment for cancer involves accurately identifying patients for risk-stratified therapy. Those with a rapid response to initial treatment may benefit from truncated treatment regimens, showing the value of accurate disease response measures. Cell-free circulating miRNAs (cimiRNAs) are highly stable in blood, are overexpressed in cancer and are quantifiable within the diagnostic laboratory. In classical Hodgkin lymphoma, plasma miR-21, miR-494, and miR-1973 are promising disease response biomarkers. Circulating miRNAs have the potential to assist clinical decision making and aid interpretation of positron emission tomography combined with computerized tomography. They can be performed at each consultation to assess disease response and detect relapse.


title: MicroRNA

text: MicroRNAs have the potential to be used as tools or targets for treatment of different cancers. The specific microRNA, miR-506 has been found to work as a tumor antagonist in several studies. A significant number of cervical cancer samples were found to have downregulated expression of miR-506. Additionally, miR-506 works to promote apoptosis of cervical cancer cells, through its direct target hedgehog pathway transcription factor, Gli3.


title: MicroRNA

text: Many miRNAs can directly target and inhibit cell cycle genes to control cell proliferation. A new strategy for tumor treatment is to inhibit tumor cell proliferation by repairing the defective miRNA pathway in tumors.


title: MicroRNA

text: Cancer is caused by the accumulation of mutations from either DNA damage or uncorrected errors in DNA replication. Defects in DNA repair cause the accumulation of mutations, which can lead to cancer. Several genes involved in DNA repair are regulated by microRNAs.


title: MicroRNA

text: Germ line mutations in DNA repair genes cause only 2–5% of colon cancer cases. However, altered expression of microRNAs, causing DNA repair deficiencies, are frequently associated with cancers and may be an important causal factor. Among 68 sporadic colon cancers with reduced expression of the DNA mismatch repair protein MLH1, most were found to be deficient due to epigenetic methylation of the CpG island of the MLH1 gene. However, up to 15% of MLH1-deficiencies in sporadic colon cancers appeared to be due to over-expression of the microRNA miR-155, which represses MLH1 expression.


title: MicroRNA

text: In 29–66% of glioblastomas, DNA repair is deficient due to epigenetic methylation of the MGMT gene, which reduces protein expression of MGMT. However, for 28% of glioblastomas, the MGMT protein is deficient, but the MGMT promoter is not methylated. In glioblastomas without methylated MGMT promoters, the level of microRNA miR-181d is inversely correlated with protein expression of MGMT and the direct target of miR-181d is the MGMT mRNA 3'UTR (the three prime untranslated region of MGMT mRNA). Thus, in 28% of glioblastomas, increased expression of miR-181d and reduced expression of DNA repair enzyme MGMT may be a causal factor.


title: MicroRNA

text: HMGA proteins (HMGA1a, HMGA1b and HMGA2) are implicated in cancer, and expression of these proteins is regulated by microRNAs. HMGA expression is almost undetectable in differentiated adult tissues, but is elevated in many cancers. HMGA proteins are polypeptides of ~100 amino acid residues characterized by a modular sequence organization. These proteins have three highly positively charged regions, termed AT hooks, that bind the minor groove of AT-rich DNA stretches in specific regions of DNA. Human neoplasias, including thyroid, prostatic, cervical, colorectal, pancreatic and ovarian carcinomas, show a strong increase of HMGA1a and HMGA1b proteins. Transgenic mice with HMGA1 targeted to lymphoid cells develop aggressive lymphoma, showing that high HMGA1 expression is associated with cancers and that HMGA1 can act as an oncogene. HMGA2 protein specifically targets the promoter of ERCC1, thus reducing expression of this DNA repair gene. ERCC1 protein expression was deficient in 100% of 47 evaluated colon cancers (though the extent to which HGMA2 was involved is not known).


title: MicroRNA

text: Single Nucleotide polymorphisms (SNPs) can alter the binding of miRNAs on 3'UTRs for example the case of hsa-mir181a and hsa-mir181b on the CDON tumor suppressor gene.


title: MicroRNA

text: The global role of miRNA function in the heart has been addressed by conditionally inhibiting miRNA maturation in the murine heart. This revealed that miRNAs play an essential role during its development. miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their involvement in cardiomyopathies. Furthermore, animal studies on specific miRNAs identified distinct roles for miRNAs both during heart development and under pathological conditions, including the regulation of key factors important for cardiogenesis, the hypertrophic growth response and cardiac conductance. Another role for miRNA in cardiovascular diseases is to use their expression levels for diagnosis, prognosis or risk stratification. miRNA's in animal models have also been linked to cholesterol metabolism and regulation.


title: MicroRNA

text: Murine microRNA-712 is a potential biomarker (i.e. predictor) for atherosclerosis, a cardiovascular disease of the arterial wall associated with lipid retention and inflammation. Non-laminar blood flow also correlates with development of atherosclerosis as mechanosenors of endothelial cells respond to the shear force of disturbed flow (d-flow). A number of pro-atherogenic genes including matrix metalloproteinases (MMPs) are upregulated by d-flow, mediating pro-inflammatory and pro-angiogenic signals. These findings were observed in ligated carotid arteries of mice to mimic the effects of d-flow. Within 24 hours, pre-existing immature miR-712 formed mature miR-712 suggesting that miR-712 is flow-sensitive. Coinciding with these results, miR-712 is also upregulated in endothelial cells exposed to naturally occurring d-flow in the greater curvature of the aortic arch.


title: MicroRNA

text: Pre-mRNA sequence of miR-712 is generated from the murine ribosomal RN45s gene at the internal transcribed spacer region 2 (ITS2). XRN1 is an exonuclease that degrades the ITS2 region during processing of RN45s. Reduction of XRN1 under d-flow" "conditions therefore leads to the accumulation of miR-712.


title: MicroRNA

text: MiR-712 targets tissue inhibitor of metalloproteinases 3 (TIMP3). TIMPs normally regulate activity of matrix metalloproteinases (MMPs) which degrade the extracellular matrix (ECM). Arterial ECM is mainly composed of collagen and elastin fibers, providing the structural support and recoil properties of arteries. These fibers play a critical role in regulation of vascular inflammation and permeability, which are important in the development of atherosclerosis. Expressed by endothelial cells, TIMP3 is the only ECM-bound TIMP. A decrease in TIMP3 expression results in an increase of ECM degradation in the presence of d-flow. Consistent with these findings, inhibition of pre-miR712 increases expression of TIMP3 in cells, even when exposed to turbulent flow.


title: MicroRNA

text: TIMP3 also decreases the expression of TNFα (a pro-inflammatory regulator) during turbulent flow.  Activity of TNFα in turbulent flow was measured by the expression of TNFα-converting enzyme (TACE) in blood. TNFα decreased if miR-712 was inhibited or TIMP3 overexpressed, suggesting that miR-712 and TIMP3 regulate TACE activity in turbulent flow conditions.


title: MicroRNA

text: Anti-miR-712 effectively suppresses d-flow-induced miR-712 expression and increases TIMP3 expression. Anti-miR-712 also inhibits vascular hyperpermeability, thereby significantly reducing atherosclerosis lesion development and immune cell infiltration.


title: MicroRNA

text: The human homolog of miR-712 was found on the RN45s homolog gene, which maintains similar miRNAs to mice. MiR-205 of humans share similar sequences with miR-712 of mice and is conserved across most vertebrates. MiR-205 and miR-712 also share more than 50% of the cell signaling targets, including TIMP3.


title: MicroRNA

text: When tested, d-flow decreased the expression of XRN1 in humans as it did in mice endothelial cells, indicating a potentially common role of XRN1 in humans.


title: MicroRNA

text: Targeted deletion of Dicer in the FoxD1-derived renal progenitor cells in a murine model resulted in a complex renal phenotype including expansion of nephron progenitors, fewer renin cells, smooth muscle arterioles, progressive mesangial loss and glomerular aneurysms. High throughput whole transcriptome profiling of the FoxD1-Dicer knockout mouse model revealed ectopic upregulation of pro-apoptotic gene, Bcl2L11 (Bim) and dysregulation of the p53 pathway with increase in p53 effector genes including Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a. p53 protein levels remained unchanged, suggesting that FoxD1 stromal miRNAs directly repress p53-effector genes. Using a lineage tracing approach followed by Fluorescent-activated cell sorting, miRNA profiling of the FoxD1-derived cells not only comprehensively defined the transcriptional landscape of miRNAs that are critical for vascular development, but also identified key miRNAs that are likely to modulate the renal phenotype in its absence. These miRNAs include miRs‐10a, 18a, 19b, 24, 30c, 92a, 106a, 130a, 152, 181a, 214, 222, 302a, 370, and 381 that regulate Bcl2L11 (Bim) and miRs‐15b, 18a, 21, 30c, 92a, 106a, 125b‐5p, 145, 214, 222, 296‐5p and 302a that regulate p53-effector genes. Consistent with the profiling results, ectopic apoptosis was observed in the cellular derivatives of the FoxD1 derived progenitor lineage and reiterates the importance of renal stromal miRNAs in cellular homeostasis.


title: MicroRNA

text: miRNAs appear to regulate the development and function of the nervous system. Neural miRNAs are involved at various stages of synaptic development, including dendritogenesis (involving miR-132, miR-134 and miR-124), synapse formation and synapse maturation (where miR-134 and miR-138 are thought to be involved). Elimination of miRNA formation in mice by experimental silencing of Dicer has led to pathological outcomes, such as reduced neuronal size and motor abnormalities when silenced in striatal neurons and neurodegeneration when silenced in forebrain neurons. Some studies find altered miRNA expression in Alzheimer's disease, as well as schizophrenia, bipolar disorder, major depression and anxiety disorders.


title: MicroRNA

text: According to the Center for Disease Control and Prevention, Stroke is one of the leading causes of death and long-term disability in America. 87% of the cases are ischemic strokes, which results from blockage in the artery of the brain that carries oxygen-rich blood. The obstruction of the blood flow means the brain cannot receive necessary nutrients, such as oxygen and glucose, and remove wastes, such as carbon dioxide. miRNAs plays a role in posttranslational gene silencing by targeting genes in the pathogenesis of cerebral ischemia, such as the inflammatory, angiogenesis, and apoptotic pathway.


title: MicroRNA

text: The vital role of miRNAs in gene expression is significant to addiction, specifically alcoholism. Chronic alcohol abuse results in persistent changes in brain function mediated in part by alterations in gene expression. miRNA global regulation of many downstream genes deems significant regarding the reorganization or synaptic connections or long term neural adaptations involving the behavioral change from alcohol consumption to withdrawal and/or dependence. Up to 35 different miRNAs have been found to be altered in the alcoholic post-mortem brain, all of which target genes that include the regulation of the cell cycle, apoptosis, cell adhesion, nervous system development and cell signaling. Altered miRNA levels were found in the medial prefrontal cortex of alcohol-dependent mice, suggesting the role of miRNA in orchestrating translational imbalances and the creation of differentially expressed proteins within an area of the brain where complex cognitive behavior and decision making likely originate.


title: MicroRNA

text: miRNAs can be either upregulated or downregulated in response to chronic alcohol use. miR-206 expression increased in the prefrontal cortex of alcohol-dependent rats, targeting the transcription factor brain-derived neurotrophic factor (BDNF) and ultimately reducing its expression. BDNF plays a critical role in the formation and maturation of new neurons and synapses, suggesting a possible implication in synapse growth/synaptic plasticity in alcohol abusers. miR-155, important in regulating alcohol-induced neuroinflammation responses, was found to be upregulated, suggesting the role of microglia and inflammatory cytokines in alcohol pathophysiology. Downregulation of miR-382 was found in the nucleus accumbens, a structure in the basal forebrain significant in regulating feelings of reward that power motivational habits. miR-382 is the target for the dopamine receptor D1 (DRD1), and its overexpression results in the upregulation of DRD1 and delta fosB, a transcription factor that activates a series of transcription events in the nucleus accumbens that ultimately result in addictive behaviors. Alternatively, overexpressing miR-382 resulted in attenuated drinking and the inhibition of DRD1 and delta fosB upregulation in rat models of alcoholism, demonstrating the possibility of using miRNA-targeted pharmaceuticals in treatments.


title: MicroRNA

text: miRNAs play crucial roles in the regulation of stem cell progenitors differentiating into adipocytes. Studies to determine what role pluripotent stem cells play in adipogenesis, were examined in the immortalized human bone marrow-derived stromal cell line hMSC-Tert20. Decreased expression of miR-155, miR-221, and miR-222, have been found during the adipogenic programming of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Conversely, ectopic expression of the miRNAs 155, 221, and 222 significantly inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/enhancer-binding protein alpha (CEBPA). This paves the way for possible genetic obesity treatments.


title: MicroRNA

text: Another class of miRNAs that regulate insulin resistance, obesity, and diabetes, is the let-7 family. Let-7 accumulates in human tissues during the course of aging. When let-7 was ectopically overexpressed to mimic accelerated aging, mice became insulin-resistant, and thus more prone to high fat diet-induced obesity and diabetes. In contrast when let-7 was inhibited by injections of let-7-specific antagomirs, mice become more insulin-sensitive and remarkably resistant to high fat diet-induced obesity and diabetes. Not only could let-7 inhibition prevent obesity and diabetes, it could also reverse and cure the condition. These experimental findings suggest that let-7 inhibition could represent a new therapy for obesity and type 2 diabetes.


title: MicroRNA

text: miRNAs also play crucial roles in the regulation of complex enzymatic cascades including the hemostatic blood coagulation system. Large scale studies of functional miRNA targeting have recently uncovered rationale therapeutic targets in the hemostatic system. They have been directly linked to Calcium homeostasis in the endoplasmic reticulum, which is critical in cell differentiation in early development.


title: MicroRNA

text: miRNAs are considered to be key regulators of many developmental, homeostatic, and immune processes in plants. Their roles in plant development include shoot apical meristem development, leaf growth, flower formation, seed production, or root expansion. In addition, they play a complex role in responses to various abiotic stresses comprising heat stress, low-temperature stress, drought stress, light stress, or gamma radiation exposure.


title: MicroRNA

text: Viral microRNAs play an important role in the regulation of gene expression of viral and/or host genes to benefit the virus. Hence, miRNAs play a key role in host–virus interactions and pathogenesis of viral diseases. The expression of transcription activators by "human herpesvirus-6" DNA is believed to be regulated by viral miRNA.


title: MicroRNA

text: miRNAs can bind to target messenger RNA (mRNA) transcripts of protein-coding genes and negatively control their translation or cause mRNA degradation. It is of key importance to identify the miRNA targets accurately. A comparison of the predictive performance of eighteen "in silico" algorithms is available. Large scale studies of functional miRNA targeting suggest that many functional miRNAs can be missed by target prediction algorithms.


title: Zebrafish

text: The zebrafish (Danio rerio) is a freshwater fish belonging to the minnow family (Cyprinidae) of the order Cypriniformes. Native to South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio (and thus often called a "tropical fish" although both tropical and subtropical). It is also found in private ponds.


title: Zebrafish

text: The zebrafish is an important and widely used vertebrate model organism in scientific research, for example in drug development, in particular pre-clinical development. It is also notable for its regenerative abilities, and has been modified by researchers to produce many transgenic strains.


title: Zebrafish

text: The zebrafish is a derived member of the genus "Brachydanio", of the family Cyprinidae. It has a sister-group relationship with "Danio aesculapii". Zebrafish are also closely related to the genus "Devario", as demonstrated by a phylogenetic tree of close species.


title: Zebrafish

text: The zebrafish is native to fresh water habitats in South Asia where it is found in India, Pakistan, Bangladesh, Nepal and Bhutan. The northern limit is in the South Himalayas, ranging from the Sutlej river basin in the Pakistan–India border region to the state of Arunachal Pradesh in northeast Indian. Its range is concentrated in the Ganges and Brahmaputra River basins, and the species was first described from Kosi River (lower Ganges basin) of India. Its range further south is more local, with scattered records from the Western and Eastern Ghats regions. It has frequently been said to occur in Myanmar (Burma), but this is entirely based on pre-1930 records and likely refers to close relatives only described later, notably "Danio kyathit". Likewise, old records from Sri Lanka are highly questionable and remain unconfirmed.


title: Zebrafish

text: Zebrafish have been introduced to California, Connecticut, Florida and New Mexico in the United States, presumably by deliberate release by aquarists or by escape from fish farms. The New Mexico population had been extirpated by 2003 and it is unclear if the others survive, as the last published records were decades ago. Elsewhere the species has been introduced to Colombia and Malaysia.


title: Zebrafish

text: Zebrafish typically inhabit moderately flowing to stagnant clear water of quite shallow depth in streams, canals, ditches, oxbow lakes, ponds and rice paddies. There is usually some vegetation, either submerged or overhanging from the banks, and the bottom is sandy, muddy or silty, often mixed with pebbles or gravel. In surveys of zebrafish locations throughout much of its Bangladeshi and Indian distribution, the water had a near-neutral to somewhat basic pH and mostly ranged from in temperature. One unusually cold site was only and another unusually warm site was , but the zebrafish still appeared healthy. The unusually cold temperature was at one of the highest known zebrafish locations at above sea level, although the species has been recorded to .


title: Zebrafish

text: The zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a zebra's stripes, and which extend to the end of the caudal fin. Its shape is fusiform and laterally compressed, with its mouth directed upwards. The male is torpedo-shaped, with gold stripes between the blue stripes; the female has a larger, whitish belly and silver stripes instead of gold. Adult females exhibit a small genital papilla in front of the anal fin origin. The zebrafish can reach up to in length, although they typically are in the wild with some variations depending on location. Its lifespan in captivity is around two to three years, although in ideal conditions, this may be extended to over five years. In the wild it is typically an annual species.


title: Zebrafish

text: In 2015, a study was published about zebrafishes' capacity for episodic memory. The individuals showed a capacity to remember context with respect to objects, locations and occasions (what, when, where). Episodic memory is a capacity of explicit memory systems, typically associated with conscious experience.


title: Zebrafish

text: The Mauthner cells integrate a wide array of sensory stimuli to produce the escape reflex. Those stimuli are found to include the lateral line signals by McHenry et al 2009 and visual signals consistent with looming objects by Temizer et al 2015, Dunn et al 2016, and Yao et al 2016.


title: Zebrafish

text: The approximate generation time for "Danio rerio" is three months. A male must be present for ovulation and spawning to occur. Zebrafish are asynchronous spawners and under optimal conditions (such as food availability and favorable water parameters) can spawn successfully frequently, even on a daily basis. Females are able to spawn at intervals of two to three days, laying hundreds of eggs in each clutch. Upon release, embryonic development begins; in absence of sperm, growth stops after the first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makes "D. rerio" a convenient research model species. Sex determination of common laboratory strains was shown to be a complex genetic trait, rather than to follow a simple ZW or XY system.


title: Zebrafish

text: The zebrafish embryo develops rapidly, with precursors to all major organs appearing within 36 hours of fertilization. The embryo begins as a yolk with a single enormous cell on top (see image, 0 h panel), which divides into two (0.75 h panel) and continues dividing until there are thousands of small cells (3.25 h panel). The cells then migrate down the sides of the yolk (8 h panel) and begin forming a head and tail (16 h panel). The tail then grows and separates from the body (24 h panel). The yolk shrinks over time because the fish uses it for food as it matures during the first few days (72 h panel). After a few months, the adult fish reaches reproductive maturity (bottom panel).


title: Zebrafish

text: To encourage the fish to spawn, some researchers use a fish tank with a sliding bottom insert, which reduces the depth of the pool to simulate the shore of a river. Zebrafish spawn best in the morning due to their Circadian rhythms. Researchers have been able to collect 10,000 embryos in 10 minutes using this method. In particular, one pair of adult fish is capable of laying 200–300 eggs in one morning in approximately 5 to 10 at time. Male zebrafish are furthermore known to respond to more pronounced markings on females, i.e., "good stripes", but in a group, males will mate with whichever females they can find. What attracts females is not currently understood. The presence of plants, even plastic plants, also apparently encourages spawning.


title: Zebrafish

text: Exposure to environmentally relevant concentrations of diisononyl phthalate (DINP), commonly used in a large variety of plastic items, disrupt the endocannabinoid system and thereby affect reproduction in a sex-specific manner.


title: Zebrafish

text: Zebrafish are omnivorous, primarily eating zooplankton, phytoplankton, insects and insect larvae, although they can eat a variety of other foods, such as worms and small crustaceans, if their preferred food sources are not readily available.


title: Zebrafish

text: Zebrafish are hardy fish and considered good for beginner aquarists. Their enduring popularity can be attributed to their playful disposition, as well as their rapid breeding, aesthetics, cheap price and broad availability. They also do well in schools or shoals of six or more, and interact well with other fish species in the aquarium. However, they are susceptible to "Oodinium" or velvet disease, microsporidia ("Pseudoloma neurophilia"), and "Mycobacterium" species. Given the opportunity, adults eat hatchlings, which may be protected by separating the two groups with a net, breeding box or separate tank.


title: Zebrafish

text: In captivity, zebrafish live approximately forty-two months. Some captive zebrafish can develop a curved spine.


title: Zebrafish

text: The zebra danio was also used to make genetically modified fish and were the first species to be sold as GloFish (fluorescent colored fish).


title: Zebrafish

text: In late 2003, transgenic zebrafish that express green, red, and yellow fluorescent proteins became commercially available in the United States. The fluorescent strains are tradenamed GloFish; other cultivated varieties include "golden", "sandy", "longfin" and "leopard".


title: Zebrafish

text: The leopard danio, previously known as "Danio frankei", is a spotted colour morph of the zebrafish which arose due to a pigment mutation. Xanthistic forms of both the zebra and leopard pattern, along with long-finned strains, have been obtained via selective breeding programs for the aquarium trade.


title: Zebrafish

text: Various transgenic and mutant strains of zebrafish were stored at the China Zebrafish Resource Center (CZRC), a non-profit organization, which was jointly supported by the Ministry of Science and Technology of China and the Chinese Academy of Sciences.


title: Zebrafish

text: The Zebrafish Information Network (ZFIN) provides up-to-date information about current known wild-type (WT) strains of "D. rerio", some of which are listed below.


title: Zebrafish

text: Hybrids between different "Danio" species may be fertile: for example, between "D. rerio" and "D. nigrofasciatus".


title: Zebrafish

text: "D. rerio" is a common and useful scientific model organism for studies of vertebrate development and gene function. Its use as a laboratory animal was pioneered by the American molecular biologist George Streisinger and his colleagues at the University of Oregon in the 1970s and 1980s; Streisinger's zebrafish clones were among the earliest successful vertebrate clones created. Its importance has been consolidated by successful large-scale forward genetic screens (commonly referred to as the Tübingen/Boston screens). The fish has a dedicated online database of genetic, genomic, and developmental information, the Zebrafish Information Network (ZFIN). The Zebrafish International Resource Center (ZIRC) is a genetic resource repository with 29,250 alleles available for distribution to the research community. "D. rerio" is also one of the few fish species to have been sent into space.


title: Zebrafish

text: Research with "D. rerio" has yielded advances in the fields of developmental biology, oncology, toxicology, reproductive studies, teratology, genetics, neurobiology, environmental sciences, stem cell research, regenerative medicine, muscular dystrophies and evolutionary theory.


title: Zebrafish

text: As a model biological system, the zebrafish possesses numerous advantages for scientists. Its genome has been fully sequenced, and it has well-understood, easily observable and testable developmental behaviors. Its embryonic development is very rapid, and its embryos are relatively large, robust, and transparent, and able to develop outside their mother. Furthermore, well-characterized mutant strains are readily available.


title: Zebrafish

text: Other advantages include the species' nearly constant size during early development, which enables simple staining techniques to be used, and the fact that its two-celled embryo can be fused into a single cell to create a homozygous embryo. The zebrafish is also demonstrably similar to mammalian models and humans in toxicity testing, and exhibits a diurnal sleep cycle with similarities to mammalian sleep behavior. However, zebrafish are not a universally ideal research model; there are a number of disadvantages to their scientific use, such as the absence of a standard diet and the presence of small but important differences between zebrafish and mammals in the roles of some genes related to human disorders.


title: Zebrafish

text: Zebrafish have the ability to regenerate their heart and lateral line hair cells during their larval stages. The cardiac regenerative process likely involves signaling pathways such as Notch and Wnt; hemodynamic changes in the damaged heart are sensed by ventricular endothelial cells and their associated cardiac cilia by way of the mechanosensitive ion channel TRPV4, subsequently facilitating the Notch signaling pathway via KLF2 and activating various downstream effectors such as BMP-2 and HER2/neu. In 2011, the British Heart Foundation ran an advertising campaign publicising its intention to study the applicability of this ability to humans, stating that it aimed to raise £50 million in research funding.


title: Zebrafish

text: Zebrafish have also been found to regenerate photoreceptor cells and retinal neurons following injury, which has been shown to be mediated by the dedifferentiation and proliferation of Müller glia. Researchers frequently amputate the dorsal and ventral tail fins and analyze their regrowth to test for mutations. It has been found that histone demethylation occurs at the site of the amputation, switching the zebrafish's cells to an "active", regenerative, stem cell-like state. In 2012, Australian scientists published a study revealing that zebrafish use a specialised protein, known as fibroblast growth factor, to ensure their spinal cords heal without glial scarring after injury. In addition, hair cells of the posterior lateral line have also been found to regenerate following damage or developmental disruption. Study of gene expression during regeneration has allowed for the identification of several important signaling pathways involved in the process, such as Wnt signaling and Fibroblast growth factor.


title: Zebrafish

text: In probing disorders of the nervous system, including neurodegenerative diseases, movement disorders, psychiatric disorders and deafness, researchers are using the zebrafish to understand how the genetic defects underlying these conditions cause functional abnormalities in the human brain, spinal cord and sensory organs. Researchers have also studied the zebrafish to gain new insights into the complexities of human musculoskeletal diseases, such as muscular dystrophy. Another focus of zebrafish research is to understand how a gene called Hedgehog, a biological signal that underlies a number of human cancers, controls cell growth.


title: Zebrafish

text: Inbred strains and traditional outbred stocks have not been developed for laboratory zebrafish, and the genetic variability of wild-type lines among institutions may contribute to the replication crisis in biomedical research. Genetic differences in wild-type lines among populations maintained at different research institutions have been demonstrated using both Single-nucleotide polymorphisms and microsatellite analysis.


title: Zebrafish

text: Due to their fast and short life cycles and relatively large clutch sizes, "D. rerio" or zebrafish are a useful model for genetic studies. A common reverse genetics technique is to reduce gene expression or modify splicing using Morpholino antisense technology. Morpholino oligonucleotides (MO) are stable, synthetic macromolecules that contain the same bases as DNA or RNA; by binding to complementary RNA sequences, they can reduce the expression of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after the 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are interpermeable to large molecules, allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies. Morpholinos are commonly microinjected in 500pL directly into 1-2 cell stage zebrafish embryos. The morpholino is able to integrate into most cells of the embryo.


title: Zebrafish

text: A known problem with gene knockdowns is that, because the genome underwent a duplication after the divergence of ray-finned fishes and lobe-finned fishes, it is not always easy to silence the activity of one of the two gene paralogs reliably due to complementation by the other paralog. Despite the complications of the zebrafish genome, a number of commercially available global platforms exist for analysis of both gene expression by microarrays and promoter regulation using ChIP-on-chip.


title: Zebrafish

text: The Wellcome Trust Sanger Institute started the zebrafish genome sequencing project in 2001, and the full genome sequence of the Tuebingen reference strain is publicly available at the National Center for Biotechnology Information (NCBI)'s Zebrafish Genome Page. The zebrafish reference genome sequence is annotated as part of the Ensembl project, and is maintained by the Genome Reference Consortium.


title: Zebrafish

text: In 2009, researchers at the Institute of Genomics and Integrative Biology in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, containing an estimated 1.7 billion genetic letters. The genome of the wild zebrafish was sequenced at 39-fold coverage. Comparative analysis with the zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations. The zebrafish reference genome sequence of 1.4GB and over 26,000 protein coding genes was published by Kerstin Howe "et al." in 2013.


title: Zebrafish

text: In October 2001, researchers from the University of Oklahoma published "D. rerio's" complete mitochondrial DNA sequence. Its length is 16,596 base pairs. This is within 100 base pairs of other related species of fish, and it is notably only 18 pairs longer than the goldfish ("Carassius auratus") and 21 longer than the carp ("Cyprinus carpio"). Its gene order and content are identical to the common vertebrate form of mitochondrial DNA. It contains 13 protein-coding genes and a noncoding control region containing the origin of replication for the heavy strand. In between a grouping of five tRNA genes, a sequence resembling vertebrate origin of light strand replication is found. It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates' nucleotide sequences.


title: Zebrafish

text: T-boxes and homeoboxes are vital in "Danio" similarly to other vertebrates. The Bruce et al team are known for this area, and in Bruce et al 2003 & Bruce et al 2005 uncover the role of two of these elements in oocytes of this species. By interfering via a dominant nonfunctional allele and a morpholino they find the T-box transcription activator Eomesodermin and its target "mtx2" – a transcription factor – are vital to epiboly. (In Bruce et al 2003 they failed to support the possibility that Eomesodermin behaves like Vegt. Neither they nor anyone else has been able to locate any mutation which – in the mother – will prevent initiation of the mesoderm or endoderm development processes in this species.)


title: Zebrafish

text: In 1999, the "nacre" mutation was identified in the zebrafish ortholog of the mammalian "MITF" transcription factor. Mutations in human "MITF" result in eye defects and loss of pigment, a type of Waardenburg Syndrome. In December 2005, a study of the "golden" strain identified the gene responsible for its unusual pigmentation as SLC24A5, a solute carrier that appeared to be required for melanin production, and confirmed its function with a Morpholino knockdown. The orthologous gene was then characterized in humans and a one base pair difference was found to strongly segregate fair-skinned Europeans and dark-skinned Africans. Zebrafish with the "nacre" mutation have since been bred with fish with a "roy orbison (roy)" mutation to make fish that have no melanophores or iridophores, and are transparent into adulthood. These fish are characterized by uniformly pigmented eyes and translucent skin.


title: Zebrafish

text: Transgenesis is a popular approach to study the function of genes in zebrafish. Construction of transgenic zebrafish is rather easy by a method using the "Tol2" transposon system. "Tol2" element which encodes a gene for a fully functional transposase capable of catalyzing transposition in the zebrafish germ lineage. "Tol2" is the only natural DNA transposable element in vertebrates from which an autonomous member has been identified. Examples include the artificial interaction produced between LEF1 and Catenin beta-1/β-catenin/"CTNNB1". Dorsky et al 2002 investigated the developmental role of Wnt by transgenically expressing a Lef1/β-catenin reporter.


title: Zebrafish

text: There are well-established protocols for editing zebrafish genes using CRISPR-Cas9 and this tool has been used to generate genetically modified models.


title: Zebrafish

text: In 2008, researchers at Boston Children's Hospital developed a new strain of zebrafish, named Casper, whose adult bodies had transparent skin. This allows for detailed visualization of cellular activity, circulation, metastasis and many other phenomena. In 2019 researchers published a crossing of a "prkdc" and a "IL2rga" strain that produced transparent, immunodeficient offspring, lacking natural killer cells as well as B- and T-cells. This strain can be adapted to warm water and the absence of an immune system makes the use of patient derived xenografts possible. In January 2013, Japanese scientists genetically modified a transparent zebrafish specimen to produce a visible glow during periods of intense brain activity.


title: Zebrafish

text: In January 2007, Chinese researchers at Fudan University genetically modified zebrafish to detect oestrogen pollution in lakes and rivers, which is linked to male infertility. The researchers cloned oestrogen-sensitive genes and injected them into the fertile eggs of zebrafish. The modified fish turned green if placed into water that was polluted by oestrogen.


title: Zebrafish

text: In 2015, researchers at Brown University discovered that 10% of zebrafish genes do not need to rely on the U2AF2 protein to initiate RNA splicing. These genes have the DNA base pairs AC and TG as repeated sequences at the ends of each intron. On the 3'ss (3' splicing site), the base pairs adenine and cytosine alternate and repeat, and on the 5'ss (5' splicing site), their complements thymine and guanine alternate and repeat as well. They found that there was less reliance on U2AF2 protein than in humans, in which the protein is required for the splicing process to occur. The pattern of repeating base pairs around introns that alters RNA secondary structure was found in other teleosts, but not in tetrapods. This indicates that an evolutionary change in tetrapods may have led to humans relying on the U2AF2 protein for RNA splicing while these genes in zebrafish undergo splicing regardless of the presence of the protein.


title: Zebrafish

text: When close relatives mate, progeny may exhibit the detrimental effects of inbreeding depression. Inbreeding depression is predominantly caused by the homozygous expression of recessive deleterious alleles. For zebrafish, inbreeding depression might be expected to be more severe in stressful environments, including those caused by anthropogenic pollution. Exposure of zebrafish to environmental stress induced by the chemical clotrimazole, an imidazole fungicide used in agriculture and in veterinary and human medicine, amplified the effects of inbreeding on key reproductive traits. Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring.


title: Zebrafish

text: Zebrafish are common models for research into fish farming, including pathogens and parasites causing yield loss and/or spread to adjacent wild populations.


title: Zebrafish

text: This usefulness is less than it might be due to "Danio"s taxonomic distance from the most common aquaculture species. Because the most common are salmonids and cod in the Protacanthopterygii and sea bass, sea bream,


title: Zebrafish

text: tilapia, and flatfish, in the Percomorpha, zebrafish results may not be perfectly applicable. Various other models Goldfish ("Carassius auratus"), Medaka ("Oryzias latipes"), Stickleback ("Gasterosteus aculeatus"), Roach ("Rutilus rutilus"), Pufferfish ("Takifugu rubripes"), Swordtail ("Xiphophorus hellerii") are less used normally but would be closer to particular target species.


title: Zebrafish

text: The only exception are the Carp (including Grass Carp, "Ctenopharyngodon idella") and Milkfish ("Chanos chanos") which are quite close, both being in the Cyprinidae. However it should also be noted that "Danio" consistently proves to be a useful model for mammals in many cases and there is dramatically more genetic distance between them than between "Danio" and any farmed fish.


title: Zebrafish

text: In a glucocorticoid receptor-defective mutant with reduced exploratory behavior, fluoxetine rescued the normal exploratory behavior. This demonstrates relationships between glucocorticoids, fluoxetine, and exploration in this fish.


title: Zebrafish

text: The zebrafish and zebrafish larva is a suitable model organism for drug discovery and development. As a vertebrate with 70% genetic homology with humans, it can be predictive of human health and disease, while its small size and fast development facilitates experiments on a larger and quicker scale than with more traditional in vivo studies, including the development of higher-throughput, automated investigative tools. As demonstrated through ongoing research programmes, the zebrafish model enables researchers not only to identify genes that might underlie human disease, but also to develop novel therapeutic agents in drug discovery programmes. Zebrafish embryos have proven to be a rapid, cost-efficient, and reliable teratology assay model.


title: Zebrafish

text: Drug screens in zebrafish can be used to identify novel classes of compounds with biological effects, or to repurpose existing drugs for novel uses; an example of the latter would be a screen which found that a commonly used statin (rosuvastatin) can suppress the growth of prostate cancer. To date, 65 small-molecule screens have been carried out and at least one has led to clinical trials. Within these screens, many technical challenges remain to be resolved, including differing rates of drug absorption resulting in levels of internal exposure that cannot be extrapolated from the water concentration, and high levels of natural variation between individual animals.


title: Zebrafish

text: To understand drug effects, the internal drug exposure is essential, as this drives the pharmacological effect. Translating experimental results from zebrafish to higher vertebrates (like humans) requires concentration-effect relationships, which can be derived from pharmacokinetic and pharmacodynamic analysis.


title: Zebrafish

text: Because of its small size, however, it is very challenging to quantify the internal drug exposure. Traditionally multiple blood samples would be drawn to characterize the drug concentration profile over time, but this technique remains to be developed. To date, only a single pharmacokinetic model for paracetamol has been developed in zebrafish larvae.


title: Zebrafish

text: Using smart data analysis methods, pathophysiological and pharmacological processes can be understood and subsequently translated to higher vertebrates, including humans. An example is the use of systems pharmacology, which is the integration of systems biology and pharmacometrics.


title: Zebrafish

text: Systems biology characterizes (part of) an organism by a mathematical description of all relevant processes. These can be for example different signal transduction pathways that upon a specific signal lead to a certain response. By quantifying these processes, their behaviour in healthy and diseased situation can be understood and predicted.


title: Zebrafish

text: Pharmacometrics uses data from preclinical experiments and clinical trials to characterize the pharmacological processes that are underlying the relation between the drug dose and its response or clinical outcome. These can be for example the drug absorption in or clearance from the body, or its interaction with the target to achieve a certain effect. By quantifying these processes, their behaviour after different doses or in different patients can be understood and predicted to new doses or patients.


title: Zebrafish

text: By integrating these two fields, systems pharmacology has the potential to improve the understanding of the interaction of the drug with the biological system by mathematical quantification and subsequent prediction to new situations, like new drugs or new organisms or patients.


title: Zebrafish

text: Using these computational methods, the previously mentioned analysis of paracetamol internal exposure in zebrafish larvae showed reasonable correlation between paracetamol clearance in zebrafish with that of higher vertebrates, including humans.


title: Zebrafish

text: Zebrafish have been used to make several transgenic models of cancer, including melanoma, leukemia, pancreatic cancer and hepatocellular carcinoma. Zebrafish expressing mutated forms of either the BRAF or NRAS oncogenes develop melanoma when placed onto a p53 deficient background. Histologically, these tumors strongly resemble the human disease, are fully transplantable, and exhibit large-scale genomic alterations. The BRAF melanoma model was utilized as a platform for two screens published in March 2011 in the journal "Nature". In one study, the model was used as a tool to understand the functional importance of genes known to be amplified and overexpressed in human melanoma. One gene, SETDB1, markedly accelerated tumor formation in the zebrafish system, demonstrating its importance as a new melanoma oncogene. This was particularly significant because SETDB1 is known to be involved in the epigenetic regulation that is increasingly appreciated to be central to tumor cell biology.


title: Zebrafish

text: In another study, an effort was made to therapeutically target the genetic program present in the tumor's origin neural crest cell using a chemical screening approach. This revealed that an inhibition of the DHODH protein (by a small molecule called leflunomide) prevented development of the neural crest stem cells which ultimately give rise to melanoma via interference with the process of transcriptional elongation. Because this approach would aim to target the "identity" of the melanoma cell rather than a single genetic mutation, leflunomide may have utility in treating human melanoma.


title: Zebrafish

text: In cardiovascular research, the zebrafish has been used to model human myocardial infarction model. The zebrafish heart completely regenerates after about 2 months of injury without any scar formation. Zebrafish is also used as a model for blood clotting, blood vessel development, and congenital heart and kidney disease.


title: Zebrafish

text: In programmes of research into acute inflammation, a major underpinning process in many diseases, researchers have established a zebrafish model of inflammation, and its resolution. This approach allows detailed study of the genetic controls of inflammation and the possibility of identifying potential new drugs.


title: Zebrafish

text: Zebrafish has been extensively used as a model organism to study vertebrate innate immunity. The innate immune system is capable of phagocytic activity by 28 to 30 h postfertilization (hpf) while adaptive immunity is not functionally mature until at least 4 weeks postfertilization.


title: Zebrafish

text: As the immune system is relatively conserved between zebrafish and humans, many human infectious diseases can be modeled in zebrafish. The transparent early life stages are well suited for "in vivo" imaging and genetic dissection of host-pathogen interactions. Zebrafish models for a wide range of bacterial, viral and parasitic pathogens have already been established; for example, the zebrafish model for tuberculosis provides fundamental insights into the mechanisms of pathogenesis of mycobacteria. Furthermore, robotic technology has been developed for high-throughput antimicrobial drug screening using zebrafish infection models.


title: Zebrafish

text: Another notable characteristic of the zebrafish is that it possesses four types of cone cell, with ultraviolet-sensitive cells supplementing the red, green and blue cone cell subtypes found in humans. Zebrafish can thus observe a very wide spectrum of colours. The species is also studied to better understand the development of the retina; in particular, how the cone cells of the retina become arranged into the so-called 'cone mosaic'. Zebrafish, in addition to certain other teleost fish, are particularly noted for having extreme precision of cone cell arrangement.


title: Zebrafish

text: This study of the zebrafish's retinal characteristics has also extrapolated into medical enquiry. In 2007, researchers at University College London grew a type of zebrafish adult stem cell found in the eyes of fish and mammals that develops into neurons in the retina. These could be injected into the eye to treat diseases that damage retinal neurons—nearly every disease of the eye, including macular degeneration, glaucoma, and diabetes-related blindness. The researchers studied Müller glial cells in the eyes of humans aged from 18 months to 91 years, and were able to develop them into all types of retinal neurons. They were also able to grow them easily in the lab. The stem cells successfully migrated into diseased rats' retinas, and took on the characteristics of the surrounding neurons. The team stated that they intended to develop the same approach in humans.


title: Zebrafish

text: Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. Zebrafish is widely used as model organism to study muscular dystrophies. For example, the "sapje" ("sap") mutant is the zebrafish orthologue of human Duchenne muscular dystrophy (DMD). The Machuca-Tzili and co-workers applied zebrafish to determine the role of alternative splicing factor, MBNL, in myotonic dystrophy type 1 (DM1) pathogenesis. More recently, Todd et al. described a new zebrafish model designed to explore the impact of CUG repeat expression during early development in DM1 disease. Zebrafish is also an excellent animal model to study congenital muscular dystrophies including CMD Type 1 A (CMD 1A) caused by mutation in the human laminin α2 (LAMA2) gene. The zebrafish, because of its advantages discussed above, and in particular the ability of zebrafish embryos to absorb chemicals, has become a model of choice in screening and testing new drugs against muscular dystrophies.


title: Zebrafish

text: Zebrafish have been used as model organisms for bone metabolism, tissue turnover, and resorbing activity. These processes are largely evolutionary conserved. They have been used to study osteogenesis (bone formation), evaluating differentiation, matrix deposition activity, and cross-talk of skeletal cells, to create and isolate mutants modeling human bone diseases, and test new chemical compounds for the ability to revert bone defects. The larvae can be used to follow new ("de novo") osteoblast formation during bone development. They start mineralising bone elements as early as 4 days post fertilisation. Recently, adult zebrafish are being used to study complex age related bone diseases such as osteoporosis and osteogenesis imperfecta. The (elasmoid) scales of zebrafish function as a protective external layer and are little bony plates made by osteoblasts. These exoskeletal structures are formed by bone matrix depositing osteoblasts and are remodeled by osteoclasts. The scales also act as the main calcium storage of the fish. They can be cultured ex-vivo (kept alive outside of the organism) in a multi-well plate, which allows manipulation with drugs and even screening for new drugs that could change bone metabolism (between osteoblasts and osteoclasts).


title: Zebrafish

text: Zebrafish pancreas development is very homologous to mammals, such as mice. The signaling mechanisms and way the pancreas functions are very similar. The pancreas has an endocrine compartment, which contains a variety of cells. Pancreatic PP cells that produce polypeptides, and β-cells that produce insulin are two examples of those such cells. This structure of the pancreas, along with the glucose homeostasis system, are helpful in studying diseases, such as diabetes, that are related to the pancreas. Models for pancreas function, such as fluorescent staining of proteins, are useful in determining the processes of glucose homeostasis and the development of the pancreas. Glucose tolerance tests have been developed using zebrafish, and can now be used to test for glucose intolerance or diabetes in humans. The function of insulin are also being tested in zebrafish, which will further contribute to human medicine. The majority of work done surrounding knowledge on glucose homeostasis has come from work on zebrafish transferred to humans.


title: Zebrafish

text: Zebrafish have been used as a model system to study obesity, with research into both genetic obesity and over-nutrition induced obesity. Obese zebrafish, similar to obese mammals, show dysregulation of lipid controlling metabolic pathways, which leads to weight gain without normal lipid metabolism. Also like mammals, zebrafish store excess lipids in visceral, intramuscular, and subcutaneous adipose deposits. These reasons and others make zebrafish good models for studying obesity in humans and other species. Genetic obesity is usually studied in transgenic or mutated zebrafish with obesogenic genes. As an example, transgenic zebrafish with overexpressed AgRP, an endogenous melacortin antagonist, showed increased body weight and adipose deposition during growth. Though zebrafish genes may not be the exact same as human genes, these tests could provide important insight into possible genetic causes and treatments for human genetic obesity. Diet-induced obesity zebrafish models are useful, as diet can be modified from a very early age. High fat diets and general overfeeding diets both show rapid increases in adipose deposition, increased BMI, hepatosteatosis, and hypertriglyceridemia. However, the normal fat, overfed specimens are still metabolically healthy, while high-fat diet specimens are not. Understanding differences between types of feeding-induced obesity could prove useful in human treatment of obesity and related health conditions.


title: Zebrafish

text: Zebrafish have been used as a model system to study epilepsy. Mammalian seizures can be recapitulated molecularly, behaviorally, and electrophysiologically, using a fraction of the resources required for experiments in mammals.


title: Filmography and awards of Sunny Leone

text: Sunny Leone is a Canadian-American actress and model, currently active in Indian film industry. She is a former pornstar. She was named "Penthouse" Pet of the Year in 2003, was a contract performer for Vivid Entertainment, and was named by "Maxim" as one of the 12 top porn stars in 2010. She has played roles in independent mainstream events, films and television shows. Her first mainstream appearance was in 2005, when she worked as a red carpet reporter for the MTV Video Music Awards on MTV India. She participated in the Indian reality television series "Bigg Boss" (2011–12). She also has been co-hosting the Indian reality TV show "Splitsvilla" (2014–present). In 2012 she made her Bollywood debut in Pooja Bhatt's erotic thriller "Jism 2" (2012) and shifted her focus to mainstream acting which was followed up with "Jackpot" (2013), "Ragini MMS 2" (2014), "Ek Paheli Leela" (2015), "One Night Stand" (2016) and "Tera Intezaar" (2017). She is known to be one of the porn stars. Sunny Leone also worked in a Bangla Cinema in an item number.


title: Acid Bath

text: Acid Bath was an American sludge metal band from Houma, Louisiana, active from 1991 to 1997. Acid Bath combined doom metal roots with influences from hardcore punk, death metal, gothic rock, and blues to create the band's unique sound. They broke up after the death of bassist Audie Pitre in a traffic collision in January 1997.


title: Acid Bath

text: Acid Bath was an incarnation of two bands, Dark Karnival which featured Audie Pitre, Sammy "Pierre" Duet, and Tommy Viator, and Golgotha which featured Dax Riggs, Mike Sanchez, Jimmy Kyle, and Jerry "Boon" Businelli. Golgotha's bassists were in frequent rotation finally culminating with Chad Pierce, who would then be succeeded by Pitre during Acid Bath's formation. Tommy Viator was later replaced by Jimmy Kyle on drums but would return to play keyboards toward the end of the band's career. Joseph J. Fontenot was the bassist for a short period of time.


title: Acid Bath

text: Acid Bath formed in 1991. The band was based south of New Orleans in several small towns including Houma, Thibodaux, Morgan City and Galliano. Influenced by thrash metal as well as bands and artists such as Black Sabbath, Alice Cooper, Celtic Frost, Carcass and Darkthrone, they recorded their demo "Hymns of the Needle Freak" with their manager and producer Keith Falgout in 1993. The strength of the demo earned them a deal with Rotten Records, an independent label. They released "When the Kite String Pops" produced by Spike Cassidy in 1994, followed two years later by their second and final effort "Paegan Terrorism Tactics" (also produced by Keith Falgout). Neither gained them mainstream success, but both albums earned a high degree of underground acclaim. In 2005, another album composed of their demos, "", was released.


title: Acid Bath

text: After two studio albums, Acid Bath's career came to an abrupt end on January 23, 1997, when bass guitarist Audie Pitre and his parents were killed by a drunk driver who had run a stop sign. Kelly Pitre, Audie's brother, was the only one of four family members to survive the incident, escaping with only a broken rib and a mild neck fracture.


title: Acid Bath

text: While rumors of another album circulated after the band's end, nothing new surfaced. Sammy Duet owns a tape of he and Mike Sanchez coming up with riffs for a possible third Acid Bath album. He mentioned this tape on his interview with "Does It Doom?". The tape was labeled "Rat Poison" because neither of them could come up with an album name. One of the members saw some rat poison in a box at the rehearsal space and said "just call it fucking Rat Poison". It was also rumored that some of the riffs were used for the first Goatwhore album; however, this was proven to be false.


title: Acid Bath

text: Tommy Viator played drums on Disincarnate's album "Dreams of the Carrion Kind" with the well-known death metal guitarist James Murphy. Dax Riggs and Mike Sanchez went on to perform in the band Agents of Oblivion, releasing one self-titled album in 2000 and disbanding shortly thereafter. Starting in 2000, Riggs was also the frontman for the swamp rock band Deadboy & the Elephantmen, before he began releasing material under his own name in 2007. Sammy Pierre Duet was once a member of Crowbar, but has since left the band. He is now a member of the blackened death metal band Goatwhore and Ritual Killer and his doom metal band with Kelly Pitre (the brother of Audie) Vual. Sammy Duet has remained an open satanist since his days in Acid Bath. Audie formed a metal band in 1995, blending black metal vocals with the heavy sound of multiple bassists with no guitars, known as Shrüm. Tommy Viator and Joseph Fontenot were also members of Shrüm. Shrüm utilized two distorted low-end basses, and Fontenot would alternate between playing a distorted high-end bass and a clean bass. Fontenot later played bass for Devourment for two years. Joseph Fontenot is now a drill sergeant in the U.S. Army.


title: Acid Bath

text: In 2014, rumors started that Acid Bath were reuniting with a new vocalist because Jimmy Kyle reached out to vocalist of Slipknot Corey Taylor with a very vague message stating "Acid Bath is in search of a vocalist. Please send Mp3, demo, videos or any music links performing Acid Bath songs." However, these rumors were dismissed by other band members stating that "There is no ACID BATH without [late bassist] Audie Pitre, so there will never be 'new' ACID BATH material", and that "Currently, most of the surviving members of ACID BATH — Jimmy, Mike, and myself — have been considering the possibility of doing some shows in the future, as an ACID BATH tribute band, but nothing has been set in stone, and it is still just an idea."


title: Acid Bath

text: Acid Bath is best known for blending extreme, death metal-influenced sludge metal with a mixture of death growls and melancholic goth/grunge-style vocals and acoustic guitar passages, as well as use of sampling and spoken word poetry. AllMusic has called the band's sound "difficult to pigeonhole" and described it as "a blend of Black Sabbath-like sludge, bluesy Southern rock, death metal, hardcore, and hints of goth and industrial". In a "Pit Magazine" interview, vocalist Dax Riggs classified their sound as "death rock". In another interview, guitarist Sammy Duet described their sound as "gothic hardcore".


title: Acid Bath

text: The band sampled sound clips from controversial movies including "Blue Velvet" and "A Clockwork Orange". Dax Riggs' vocals were processed, which produced an industrial feel; some other instruments have been processed through industrial effects in their recordings (such as the snare drum on the second half of "New Death Sensation"). One of Acid Bath's most trademark sounds was the way in which they crafted multiple tempo changes and worked them into the song structure. Their experimentation drifted into diverse territory. The song "Scream of the Butterfly" is an acoustic blues song with the drummer with double bass drum patterns toward the end of the song. The song "The Bones of Baby Dolls" experiments with folk musicianship, and the song "Dead Girl" was described as a country song by Dax Riggs.


title: Acid Bath

text: Dax Riggs' lyrics are frequently poetic, often displaying an obsession with death, drug use, mental illness, dark humor, Louisiana-based regional culture, and continuous references to animism as well as paganism, nihilism, and misanthropy. He has claimed these inspirations are culled from comic books, namely those authored by Frank Miller, Alan Moore, and Clive Barker, and has also expressed admiration for "ANSWER Me!" and "Boiled Angel". AllMusic's William York has stated that the song "Venus Blue" could have been a radio hit "if not for the graphic lyrics". Another facet of their presentation which may not have endeared them to popular sentiment was the use of art by John Wayne Gacy and Dr. Jack Kevorkian. Due to the controversy surrounding Kevorkian's artwork "For He Is Raised" on the album, "Paegan Terrorism Tactics" was initially banned from Australia. The ban has since been lifted. The cover of the band's 1994 extended play simply entitled "Edits" featured the artwork of Richard Ramirez. The cover of their 1996 compilation entitled "Radio Edits" featured the artwork of Kenneth Bianchi. Their song "Diäb Soulé" ("Drunken Devil" in Cajun-French) starts out with an audio sample of Jim Jones of the People's Temple screaming. Their song "Toubabo Koomi" is Cajun-French for the "Land of White Cannibals".


title: Claudio Pizarro

text: Claudio Miguel Pizarro Bosio (; born 3 October 1978) is a Peruvian retired professional footballer who played as a striker. He is currently serving as Club Ambassador for Bayern Munich. He was captain of Peru's national football team, being its fifth highest scorer. He is widely considered as the greatest representative of Peruvian football in Europe. He is the highest scorer and most successful Latin American football player in the history of German football. He is the all-time top scorer of SV Werder Bremen, the ninth top scorer in the history of Bayern Munich and the sixth top scorer in the history of the Bundesliga and its second top scorer in the 21st century. He is also among the 20 top scorers in the history of UEFA club competitions and is the seventh highest South American scorer in European football history.


title: Claudio Pizarro

text: His professional debut came in 1996, with Deportivo Pesquero. In 1998, he joined Alianza Lima, where he won a Peruvian Primera División, being highlighted as the best player of the season. In 1999, he was signed by Werder Bremen from the Bundesliga. Just one year later, he was chosen as the newcomer player of the championship and was included in the ideal team of the 2000–01 season. In 2001, he signed for Bayern Munich and the following seasons he won one Intercontinental Cup, 2 Bundesligas, 4 DFB-Pokale (German Cups), 2 DFL-Ligapokale (German League Cups) and the DFL-Supercup (German Super Cup). In addition, he was the top scorer in the DFB-Pokal in the 2004–05 and 2005–06 editions.


title: Claudio Pizarro

text: In 2007, he signed for Chelsea F.C. scoring on his debut and achieving runner-up in the Premier League at the end of the season. In 2009, he returned to Bremen, where he was champion of the 2008–09 DFB-Pokal and the unofficial 2009 German Supercup, in addition to becoming top scorer in the 2009–10 UEFA Europa League. In 2012, he was hired again by Bayern Munich, winning the UEFA Champions League, the Club World Cup, the European Super Cup, the DFL-Supercup, three Bundesligas and two DFB-Pokale, thus becoming the foreign player with most titles in the history of Bayern Munich, a total of 18. In mid-2015, he returned to Werder Bremen. In 2017, he was hired by the 1. FC Köln, from which he separated a year later to return to Werder Bremen.


title: Claudio Pizarro

text: Pizarro is the Peruvian football player with the most goals scored and games played in Europe and in German football. He is the second highest foreign scorer and sixth all-time historic scorer in the Bundesliga (197 goals). He is also the Latin American football player with the most goals scored and titles won in the history of German football. He is the ninth all-time top scorer for the DFB-Pokal (34 goals), top scorer for Werder Bremen (153 goals), the ninth all-time top scorer for Bayern Munich (125 goals), the tenth historical scorer of the UEFA Europa League (24 goals) and the fifth historical scorer of Peru's national football team (20 goals). He is also the foreign player with the most games played in the history of Bundesliga and the DFB-Pokal. Pizarro is considered an idol by Bayern Munich's fans and a legend of Werder Bremen's, teams for which he scored more than 100 goals each. As well as being one of the players with the most seasons played in the history of the German league, Pizarro is also the oldest player to score a goal in the Bundesliga (at over 40 years old). He is regarded as a living legend of the Bundesliga, being compared to other German football legends like Franz Beckenbauer and Gerd Müller.


title: Claudio Pizarro

text: In international competitions, he is the fourth highest Latin American scorer in European Cups (Champions League, UEFA Cup and UEFA Europa League) with 48 goals, ranking only behind Lionel Messi, Alfredo Di Stéfano and Sergio Agüero.


title: Claudio Pizarro

text: With the Peru national football team he is the fifth highest scorer with 20 goals and the tenth with the most matches (85). With the senior team, he achieved the third place at the 2015 Copa América. He also participated in the 2004 and 2007 Copa América.


title: Claudio Pizarro

text: Pizarro was born to Patricia Bosio and Claudio Pizarro Dávila, a naval officer, in Callao, Peru and raised in Santiago de Surco district of Lima, Peru. He started playing in his youth in the Academia Deportiva Cantolao in Callao. Pizarro has Italian ancestry; some of his paternal great-grandparents were from Brescia, while his maternal grandparents came from Frattaminore, near Naples.


title: Claudio Pizarro

text: Pizarro started his professional career playing with Deportivo Pesquero, a small provincial team in the city of Chimbote in the north of Peru, at the age of 17. His first professional match was in 1996 against Alianza Lima. He scored his first two goals two weeks later against Atletico Torino in the 2–1 victory of Deportivo Pesquero. He finished the 1996 season with three goals in 16 appearances.


title: Claudio Pizarro

text: Pizarro scored 25 goals over two seasons with Alianza Lima and was part of the team that ended in second position in the Torneo Apertura of Peru in 1999. He also made seven appearances in Copa Libertadores. Shortly after his success at Alianza, Pizarro was sold to Werder Bremen in the German Bundesliga. Later that year, he earned his first call for the Peru national football team.


title: Claudio Pizarro

text: Pizarro joined Werder Bremen for the first time in the summer of 1999 when he was only 20 years old. His first match in the Bundesliga was on 28 August of that year, a 1–1 draw against Hertha BSC, joining as a second-half substitute. In his second appearance on 12 September, Pizarro scored his first Bundesliga goal in the 5–0 victory over 1. FC Kaiserslautern at home. One week later, Pizarro made his first hat-trick in the 7–2 victory over VfL Wolfsburg, establishing himself as one of the most promising talents. He ended his first Bundesliga season scoring 10 goals in 25 matches. In that season, Pizarro also made his UEFA Cup debut, scoring 3 times in 9 appearances. In his first ever match on 15 September, he scored 2 goals in the 5–0 home win over Norway's FK Bodø/Glimt. He was protagonist in the third round tie against French rivalOlympique Lyonnais. After losing the first leg away 3–0, Pizarro helped Werder Bremen on one of the best comebacks in UEFA competitions history. In the returning leg, he scored the fourth goal of a 4–0 win at Weserstadion. The result allowed Werder Bremen to move to the next round with a 4–3 on the aggregate.


title: Claudio Pizarro

text: Pizarro's 38 goals over 2 seasons for Werder Bremen made him the rising star of Peruvian football, coveted by managers of major European teams. After the 2000–01 season, Werder Bremen announced that it would not prevent Pizarro from moving out as the push to secure Pizarro's services intensified. The media indicated that Real Madrid and Barcelona, Inter Milan, and Werder Bremen's Bundesliga rival Borussia Dortmund were in to sign Pizarro. The most speculated transfer of a Peruvian footballer monopolized the interest of its local media. After seeming destined for Spain, Pizarro ended weeks of speculation by signing for Bayern Munich on 7 June 2001.


title: Claudio Pizarro

text: Pizarro started at Bayern with a goal in the fourth minute of the game against Schalke 04 in the second match of the 2001–02 Bundesliga season. In his first season, he scored 15 goals in 30 Bundesliga and 4 German Cup appearances, 4 goals in 14 appearances in the UEFA Champions League, one appearance in the Intercontinental Cup, and one appearance in the UEFA Super Cup.


title: Claudio Pizarro

text: Pizarro was a key player in Bayern' Munich's Bundesliga and DFB-Pokal double in the 2002–03 season. On 30 November 2002, Pizarro appeared in his 100th Bundesliga match in the 2–0 home win over Hertha BSC, thanks to a Michael Ballack brace. During the 2002–03 season, Pizarro scored 15 goals in 31 appearances in the Bundesliga, 2 goals in 6 DFB-Pokal appearances, 2 goals in 7 Champions League appearances. He also had 2 League Cup appearances.


title: Claudio Pizarro

text: During the 2003–04 season, he scored 11 goals in 31 Bundesliga appearances and 1 goal in 4 DFB-Pokal appearances. He also had 7 Champions League and 1 League Cup appearances.


title: Claudio Pizarro

text: During the 2005–06 season, he scored 11 goals in 26 Bundesliga appearances, 5 goals in 5 German Cup appearances, 1 goal in 6 Champions League appearances, and an appearance in the League Cup.


title: Claudio Pizarro

text: His popularity with Bayern fans earned him the nickname "Bomber of the Andes", a reference to his Peruvian origin as well as to the legendary Bayern's striker Gerd Müller, nicknamed "Bomber". On some occasions, he was also called "Inca God" by the media.


title: Claudio Pizarro

text: Pizarro's contract with the German champions expired at the end of the 2006–07 season and negotiations on an extension were not making progress, with Sevilla, Benfica and Rangers ready to step in. Pizarro later rejected a contract extension offered by Bayern Munich's director Franz Beckenbauer. Pizarro demanded an increase in pay, which angered Karl-Heinz Rummenigge, who was quoted saying that "anyone wishing to earn as much as Shevchenko had better start playing like Shevchenko".


title: Claudio Pizarro

text: On 20 May 2007, Bayern Munich announced Pizarro would be leaving the club. He finished the 2006–07 season with 12 goals in 45 appearances.


title: Claudio Pizarro

text: On 1 July 2007, Chelsea officially signed Pizarro and confirmed that he had completed a medical evaluation and agreed on personal terms to join based on a Bosman transfer, signing a four-year contract. He was the first Peruvian to sign for Chelsea. Pizarro also revealed that his decision was influenced by the advice of his national teammate Nolberto Solano, who played in England for Newcastle, Aston Villa and West Ham United, as well as Owen Hargreaves, who at the time played for Bayern Munich with Pizarro.


title: Claudio Pizarro

text: Pizarro was assigned the number 14, the same number he has in the Peruvian National Team, and his former number at Bayern Munich, succeeding Geremi at Chelsea. His debut was against Manchester United in the 2007 FA Community Shield, coming on as a substitute, but then missed the penalty shootout as Chelsea lost the match. Nevertheless, his season with Chelsea got off to a good start, scoring a goal on the opening weekend of the 2007–08 Premier League season against Birmingham City. After Chelsea's coach José Mourinho's departure and the signing of French striker Nicolas Anelka, Pizarro found himself surplus to the then new manager Avram Grant. Despite this, Pizarro did play an important role in Chelsea progressing to the fourth round of the 2008 FA Cup, by contributing to a rebound of QPR keeper Lee Camp that went into the net. He also scored the only goal in a match against Birmingham. This meant that his only two Premier League goals with Chelsea came in separate games against Birmingham City.


title: Claudio Pizarro

text: On 15 August 2008, Pizarro was loaned to Werder Bremen until the end of the season . Soon after the announcement, a large group of Werder Bremen fans lined up to buy the new Pizarro's jersey showing that he was still appreciated in Bremen seven years after leaving. He was assigned the number 24 and scored his first goal in his second appearance, Werder Bremen's first goal in a 3–2 defeat to Borussia Mönchengladbach on 30 August . Pizarro scored Werder Bremen's fourth goal in the club's impressive 5–2 away victory over Bayern Munich on 20 September at the Allianz Arena. Former Werder Bremen's teammate Tim Borowski scored both of Bayern Munich's goals.


title: Claudio Pizarro

text: On 26 February 2009, Pizarro scored a second-half brace for Werder Bremen, coming back from 2–0 down to secure a 2–2 draw and a 3–3 aggregate win (away goals rule), to dump A.C. Milan out of the UEFA Cup in the Round of 32. In the second-leg of their UEFA Cup quarter-final match against Udinese on 17 April, Pizarro scored once and teammate Diego notched a brace as the teams played out an exciting 3–3 draw, with Werder Bremen advancing to the semi-finals with a 6–4 aggregate victory. He scored a hat-trick on 4 May as Bremen beat Hannover 4–1, keeping alive their push for a European place.


title: Claudio Pizarro

text: Pizarro's role was fundamental in the qualification of Werder Bremen to the UEFA Cup Final, scoring a goal in the 3–2 away victory over Hamburg on 7 May, completing a 3–3 aggregate victory over their German rivals in the semi-finals. However, with the absence of playmaker Diego in the final against Shakhtar Donetsk, Pizarro could not save the team from a 2–1 defeat after extra time to the Ukrainian champions on 20 May 2009 at the Şükrü Saracoğlu Stadium in Istanbul.


title: Claudio Pizarro

text: Pizarro was decisive and important for Werder Bremen during his return, scoring 17 goals in just 26 Bundesliga appearances in the 2008–09 season.


title: Claudio Pizarro

text: On 18 August 2009, Pizarro signed permanently his return to Werder Bremen for an undisclosed amount after an impressing performance in the previous season. In his second season with Werder Bremen, he continued to be one of the top Bundesliga scorers with 16 goals. On 23 October 2010, Pizarro scored his 134th goal in the Bundesliga, making him the top foreign-born scorer in the league's history, after being tied with Giovane Élber. Pizarro finished the 2010–11 season with 14 goals in 29 appearances. On 15 May 2012, Pizarro announced that he would leave Werder Bremen in the summer. He finished his final season with 18 goals in 29 appearances.


title: Claudio Pizarro

text: On 26 May 2012, Pizarro signed a one-year contract with his former German team, Bayern Munich. On 31 October, Pizarro scored 2 goals against Kaiserslautern in the DFB-Pokal. In the Champions League match against Lille on 7 November, Pizarro scored a hat-trick within the first 33 minutes of the game as Bayern defeated their French opponents 6–1. On 30 March 2013, Pizarro scored 4 goals, his firsts in the Bundesliga season, and provided two assists in a 9–2 win of Bayern Munich against Hamburg. On 10 April 2013, Pizarro came off the bench to score a 90th-minute goal against Juventus in the quarter-final of the 2012–13 UEFA Champions League, which would end in a 0–2 win for Bayern Munich (0–4 in the aggregate). Pizarro scored twice more and provided a further two assists in Bayern Munich's 6–1 victory over Hannover 96 on 20 April. He finished the 2012–13 season with 13 goals in 28 appearances.


title: Claudio Pizarro

text: On 19 April 2014, Pizarro scored in Bayern Munich's 2–0 win over Eintracht Braunschweig, making him a scorer to every single Bundesliga team. During the 2013–14 season, he scored 10 goals in 17 Bundesliga appearances, 1 goal in 2 German Cup appearances. He also had 6 Champions League appearances, a German Super Cup appearance, and a FIFA World Club Cup appearance.


title: Claudio Pizarro

text: His contract wasn't renewed after the 2014–15 season, when he scored just one goal in 17 appearances. He failed to score in any of his Bundesliga or Champions League matches during the rest of the season.


title: Claudio Pizarro

text: On 7 September 2015, Pizarro joined Werder Bremen for the fourth time in his career, agreeing to a one-year contract. He was given the number 14. On 2 March 2016 Pizarro reached 10 goals in the season by scoring a hat-trick in the 4–1 win away to Bayer Leverkusen. The then 37-year old Pizarro broke a record which stood for 31 years, becoming the oldest player to score a hat-trick in the Bundesliga. He finished the 2015–16 season. On 2 July 2017, Werder Bremen announced Pizarro's contract would not be renewed. He finished the 2016–17 season with a goal in 19 appearances.


title: Claudio Pizarro

text: On 29 September 2017, Pizarro signed a one-year contract with 1. FC Köln. They were relegated from the Bundesliga following defeat to SC Freiburg on 28 April 2018. He finished the 2017–18 season with a goal in 16 appearances.


title: Claudio Pizarro

text: On 29 July 2018, Pizarro signed a one-year contract with Werder Bremen joining them for the fifth time in his career.


title: Claudio Pizarro

text: On 16 February 2019, with his stoppage equaliser in the away match against Hertha BSC, Pizarro became the oldest scorer in the history of the Bundesliga at 40 years and 136 days of age. Pizarro broke the record previously held since August 1996 by Miroslav Votava ( then 40 years and 121 days of age). His goal also made him the first and only player to score in 21 consecutive calendar years in the Bundesliga. On 4 May 2019, he scored his fourth Bundesliga goal of the season, in a 2–2 equalizer of Werder Bremen against Borussia Dortmund. His fifth goal determined the 2–1 win against RB Leipzig, raising the record for the oldest scorer to 40 years 227 days of age.


title: Claudio Pizarro

text: On 20 July 2019, Pizarro announced that the 2019–20 season would be his last season before retiring from football activity. In that season, he played 18 Bundesliga matches without scoring a goal. In all of these, he entered as substitute, including his final appearance against Mainz 05 on 27 June 2020. He was no longer considered in the team for the following 2 relegation playoff games. In his career at the Bundesliga, Pizarro scored 197 goals in 490 matches.


title: Claudio Pizarro

text: Pizarro has been a regular for Peru since scoring in a 2–1 defeat against Ecuador on his debut in February 1999. In the same year, he was part of the Peruvian team at the 1999 Copa América in Paraguay, starting all three of the group matches and coming on as a substitute in the quarter-final match, which ended with a penalty shootout loss against Mexico. Pizarro has gone on to be a starting player at the 2004, 2007 and 2015 Copa América tournaments, as well as the FIFA World Cup qualifying campaigns for the 2002, 2006, 2010, 2014 and 2018.


title: Claudio Pizarro

text: On 23 August 2003, Pizarro scored the fastest ever goal in the history of the Peruvian national team, at 18 seconds of the match's start for a 3–1 win over Mexico at Giants Stadium in New Jersey. He was later shown a red card along with teammate John Galliquio and Mexicans Jared Borgetti and Omar Briceño.


title: Claudio Pizarro

text: At the 2004 Copa América, Pizarro suffered a fractured skull when he took an elbow to the side of his head in an on-field collision with the opponent's goalkeeper during a 3–1 win over Venezuela. The injury forced him out of the rest of the tournament and required extensive surgery in Germany. Under the management of Julio César Uribe, Pizarro captained Peru to the quarter-finals of the 2007 Copa América, scoring the two goals in the 2–2 draw against Bolivia in the final match of the group in Mérida.


title: Claudio Pizarro

text: On 18 June 2015, Pizarro scored the only goal of Peru's 1–0 win against Venezuela in the group stage of the 2015 Copa América. He won his last cap in March 2016.


title: Claudio Pizarro

text: On 7 December 2007, an investigation was launched on women and alcohol found in the national squad's concentration hotel, two days before Peru's away 5–1 defeat by Ecuador. Pizarro was suspended 18 months from the national team starting on the day before the match. Other Peruvian players playing abroad, like Jefferson Farfán were also suspended. Since Pizarro claimed to be innocent, he started a trial against the Peruvian Football Federation. This action alerted FIFA about his case, who warned Pizarro not to take it to regular courts of justice or face an international sanction. On 3 July 2008, after an investigation and a review of the facts, the suspension was reduced to 3 months (from the date of appeal in April 2008) and a US$10,000 fine. Pizarro served the three-month sanction.


title: Claudio Pizarro

text: On 17 April 2009, Pizarro won a court victory over his national federation. The Court of Arbitration for Sport overturned the $10,000 fine imposed by the Peruvian Football Association after the alleged November 2007 incident. The sentence quoted: "The facts put forward by (the federation) in relation to Pizarro were not supported by concrete evidence", sport's highest court said in a statement. Pizarro expressed his rejoice with the final result, declaring: "I am very satisfied and feel vindicated". He further said. "My honor has been restored. My family and friends have always stood by me and knew that these were all just lies that were being spread about me. But this verdict will now also give certainty to everyone who had doubts that I have always told the truth."<ref name="cnn/si"></ref>


title: Claudio Pizarro

text: Pizarro has a sister, Patricia, and a younger brother, Diego. Diego is also a professional footballer and played for Bayern Munich as a youth.


title: Claudio Pizarro

text: Pizarro is married to his teenage sweetheart, Karla Salcedo, having two sons and a daughter. All three were born in Germany. Pizarro co-owns a race horse called 'Crying Lightning' with fellow professional footballer Joey Barton. In January 2011, the horse competed at the Nad Al Sheba Racecourse in Dubai.


title: Pendle witches

text: The trials of the Pendle witches in 1612 are among the most famous witch trials in English history, and some of the best recorded of the 17th century. The twelve accused lived in the area surrounding Pendle Hill in Lancashire, and were charged with the murders of ten people by the use of witchcraft. All but two were tried at Lancaster Assizes on 18–19 August 1612, along with the Samlesbury witches and others, in a series of trials that have become known as the Lancashire witch trials. One was tried at York Assizes on 27 July 1612, and another died in prison. Of the eleven who went to trial – nine women and two men – ten were found guilty and executed by hanging; one was found not guilty.


title: Pendle witches

text: The official publication of the proceedings by the clerk to the court, Thomas Potts, in his "The Wonderfull Discoverie of Witches in the Countie of Lancaster", and the number of witches hanged together – nine at Lancaster and one at York – make the trials unusual for England at that time. It has been estimated that all the English witch trials between the early 15th and early 18th centuries resulted in fewer than 500 executions; this series of trials accounts for more than two per cent of that total.


title: Pendle witches

text: Six of the Pendle witches came from one of two families, each at the time headed by a woman in her eighties: Elizabeth Southerns (a.k.a. Demdike), her daughter Elizabeth Device, and her grandchildren James and Alizon Device; Anne Whittle (a.k.a. Chattox), and her daughter Anne Redferne. The others accused were Jane Bulcock and her son John Bulcock, Alice Nutter, Katherine Hewitt, Alice Grey, and Jennet Preston. The outbreaks of 'witchcraft' in and around Pendle may suggest that some people made a living as traditional healers, using a mixture of herbal medicine and talismans or charms, which might leave them open to charges of sorcery. Many of the allegations resulted from accusations that members of the Demdike and Chattox families made against each other, perhaps because they were in competition, both trying to make a living from healing, begging, and extortion.