3,050 Matching Annotations
  1. Jan 2018
    1. Original publication date 11/01/1911 Reference 11

      THIS IS ME ANNOTATING.

      This is me looking at my annotation as I annotate.

    1. P. F. A. Maderson, When? Why? and How?: Some speculations on evolution of vertebrate integument. Am. Zool. 12, 159–171 (1972).

      A 1970's review discussing the formation of integuments (hard, protective layers covering the body), scales, and hair.

      The paper explores the different hypotheses of the time, including scaled animals as precursors to nonscaled animals, ossified (bony) scales as a precursor to the scales found on today's reptiles, and the evolution of hair from sensory appendages.

    2. A. M. Turing, The chemical basis of morphogenesis. Philos. Trans. R. Soc. London Ser. B 237, 37–72 (1952).

      In this landmark paper, Turing proposes the reaction diffusion theory to explain morphogenesis.

      This theory states that when two chemicals react with each other they diffuse to form chemical gradients. These gradients form patterns that direct morphogenesis.

    3. Both models assume that the development of an anatomical placode and of a dermal papilla occurred, at a minimum, twice (once in birds and once in mammals) through the independent parallel co-option of the same set of signaling pathways (WNTs, β-catenin, EDAR, BMPs, and SHH).

      Previous models have tried to explain the apparent lack of homology by suggesting that anatomical placodes and dermal papillae developed at least twice (once in birds and once in mammals).

      Further, the models suggest that, although the anatomical placodes and dermal papillae in mammals and birds would have evolved independently, they would have came about through the co-option of the same set of signaling pathways.

    4. integument

      A tough outer protective layer.

    5. generates a new splice donor site (gt) 42 bases upstream of the wild-type donor site, thus generating a 14–amino acid deletion in the corresponding transcript (Fig. 3D).

      The authors found that the scaleless mutation is caused by the deletion of 14 amino acids in the EDF protein.

      Their analysis indicates that the deletion is caused by the insertion of a 688-base-pair fragment which changes the way the gene is spliced (a process by which noncoding sequences are removed from mRNA before a protein is transcribed).

    6. cDNA

      DNA synthesized from single-stranded RNA.

      .

    7. ectodermal dysplasia syndrome

      A genetic disease affecting the growth of hair, teeth, nails, and sweat glands.

    8. caudal, spinal, cervical, ventral, humeral, and femoral

      Caudal: pertaining to the tail

      Spinal: pertaining to the spine

      Cervical: pertaining to the neck

      Ventral: pertaining to the underside or abdominal part of the body

      Humeral: pertaining to the humerus (a bone in the arm)

      Femoral: pertaining to the femur (a bone in the leg)

    9. whole-mount in situ hybridization (WMISH) with species-specific probes, we show that crocodile, lizard, and snake placodes all exhibit spatial expression of Shh

      WMISH is a common technique for visualizing the location of expressed RNAs in embryos.

      In this study, the authors used WMISH to show that Shh was expressed in the placode.

    10. in-frame deletion

      Because an RNA sequence is read three bases at a time, if the number of bases deleted is a multiple of three, it will not change the reading frame.

    11. material cracking

      Crocodilian scales are formed through exertion of force: Scale tissues are physically cracked by living materials, leading to unique patterns on each reptile's face.

    12. These results reveal a new evolutionary scenario where hairs, feathers, and scales of extant species are homologous structures inherited, with modification, from their shared reptilian ancestor’s skin appendages already characterized by an anatomical placode and associated signaling molecules.

      The authors found that the placode and the placode's signaling molecules are highly conserved—meaning they are found across many species. This study shows that the placode is responsible for the formation of many different structures such as feathers, scales, and hair.

    13. anatomical placode

      A thick, platelike structure in the ectoderm that is a site of development in early embryos.

    14. squamates

      Members of the order squamata, or scaled reptiles. They are the largest and most recent order of reptiles and comprise all lizards and snakes.

    15. we show for the first time

      To read a popular science version of this paper, check out Motherboard's article, which includes quotes from the author.

      http://motherboard.vice.com/read/hairy-humans-and-scaly-lizards-have-more-in-common-than-you-think

    16. placode

      A thickening of the ectoderm marking a site of future development of hair follicles, feathers, or teeth in the early embryo.

    17. fossil intermediate

      A transitional fossil that shows a transitional form between one species and another.

    18. These results indicate that all three characteristics (epidermal thickening, expression of epidermal placode markers, and expression of dermal Bmp4), that is, the presence of an anatomical placode, are required for proper development of scales in reptiles.

      Epidermal thickening, patterning of markers, and expression of dermal Bmp4, all characteristics of an anatomical placode, are necessary for reptiles to properly develop scales.

    19. R. B. Widelitz, T.-X. Jiang, J. Lu, C.-M. Chuong, b-catenin in epithelial morphogenesis: Conversion of part of avian foot scales into feather buds with a mutated b-catenin. Dev. Biol. 219, 98–114 (2000).

      Explores the role of β-catenin, which is expressed in the placode.

      When this protein was mutated in chickens, their feet were scaleless and they had abnormal feather growth.

    20. C. Blanpain, E. Fuchs, Epidermal stem cells of the skin. Annu. Rev. Cell Dev. Biol. 22, 339–373 (2006).

      Reviews the development of skin cells that give rise to hairs.

      A hair placode forms, allowing the expression of genes that direct skin cell and hair follicle development.

    21. J. M. Musser, G. P. Wagner, R. O. Prum, Nuclear b-catenin localization supports homology of feathers, avian scutate scales, and alligator scales in early development. Evol. Dev. 17, 185–194 (2015).

      Explores the relationship between feathers and scales.

      Visualization of the location and amount of β-catenin during the development of feathers and scales revealed similar patterns between the two. The authors concluded that feathers and scales share an evolutionary ancestor.

    22. D. Dhouailly, A new scenario for the evolutionary origin of hair, feather, and avian scales. J. Anat. 214, 587–606 (2009).

      Proposed the theory that mammalian hair evolved from glandular structures, whereas reptile and bird skin evolved from a thick protective coating.

    23. P. F. A.Maderson,Mammalian skin evolution: A reevaluation. Exp. Dermatol. 12, 233–236 (2003).

      A revision to a 1972 model of how mammalian hair evolved.

      This new model proposes that the development of hair was caused by mutations in patterning genes, which resulted in hair becoming more useful insulation.

    24. M. C. Milinkovitch, L. Manukyan, A. Debry, N. Di-Poï, S. Martin, D. Singh, D. Lambert, M. Zwicker, Crocodile head scales are not developmental units but emerge from physical cracking. Science 339, 78–81 (2013).

      This paper discusses the way that crocodile head scales form. Unlike feathers, hair, and other scales, crocodile facial scales are formed when growing cells physically crack to form unique patterns.

    25. It has been previously hypothesized (47) that reptilian scales are more similar to avian reticulate scales (covering the foot pad) than to both avian scutate scales (covering the anterior metatarsal region) and feathers

      It was previously thought that reptilian scales are more similar to some types of avian scales (such as those covering the foot pad) than others. However, the current study suggests that this is not the case.

      All avian scales develop from an anatomical placode similar to reptile scales.

    26. The squamate anatomical placode, whose existence had remained undetected because of its transitory developmental dynamic, exhibits all the major features characterizing avian and mammalian placodes: local epidermal thickening with columnar cells and reduced proliferation rate, shared early spatially restricted expression of epidermal molecular markers, and localized conserved signaling in the underlying dermis

      The major finding of this paper is that reptile scales develop from an anatomical placode, similar to mammalian hair and avian feathers.

      Scientists were previously unsure if this was the case because the reptilian anatomical placode is very short-lived during development (and thus hard to detect).

    27. The data presented here put this debate to rest by demonstrating that most skin appendages in amniotes, including snake and lizard overlapping scales, not only share signaling pathways during morphogenesis but also truly develop from anatomical placodes.

      The results here support the idea that most skin appendages in reptiles, mammals, and birds are homologous because they share signaling pathways and arise from an anatomical placode.

    28. Conversely, other authors argue that the similarities in molecular signaling observed among all skin appendages suffice to support their homology (9). Note that such placodal signaling centers have been recently evidenced as underlying the development of Chelonia shell scutes

      Some other models reject the idea that the placode and the dermal papilla evolved independently, suggesting that the similarities in the molecular signaling involved in development are sufficient to show that they are homologous (meaning the pathway evolved once, in a common ancestor).

      It is also suggested that this is true for turtles, which have been found to need the placode for the development of their shell.

    29. sebocytes

      A cell that secretes sebum, an oily substance that waterproofs and lubricates skin.

    30. The fossil record lacks any evidence of intermediate forms (hence, of homology) between scales and hairs.

      One of the major challenges of tracing an organism's evolutionary history is an incomplete fossil record. Where there is no intermediate between two forms, scientists have to use the available evidence to make predictions about what this intermediate would have looked like.

    31. keratinized

      To change to a form that contains keratine, a fibrous protein found in hair, nails, and hooves.

    32. Similarly, Ctnnb1 andEdar, two other placode markers, also show marked differences in expression between wild-type and scaleless dragons. In both phenotypes, expression of these two genes is first ubiquitous across the whole epidermis before becoming restricted to the placodes in wild-type individuals only (Fig. 4B). These results indicate that expression of each of these three placode markers in reptiles is similar to the expression dynamic of the corresponding genes in mammals (27) and birds (20, 28,39). On the other hand, the absence of an anatomical placode in scaleless dragons coincides with the inability of signaling pathways to pattern the skin, similar to what is observed in mice deficient in Eda/Edar(40).

      The authors demonstrate that Shh, Ctnnb1, and Edar expression is similar in reptiles, mammals, and birds. They also conclude that the absence of an anatomical placode in scaleless lizards disrupts these pathways' ability to pattern the skin, which has also been observed in mice (a mammal) with similar mutations.

    33. breeding experiments

      Individual lizards with different physical traits were bred together in order to observe how those traits were passed on to the offspring.

    34. cryosections

      Sections of tissue that are made in a cryostat, a device that keeps samples at a very low temperature to preserve them.

    35. BMP

      A group of signaling molecules responsible for coordinating development in many different parts of the body.

    36. each of these dermoepidermal elevations that generate scales in crocodiles, lizards, and snakes occurs at the location of a transient developmental unit that exhibits the characteristics (Fig. 1B) of the mammalian and avian anatomical placode

      The authors of this study expand on the findings of previous studies by showing that the elevations that result in scales correspond to the anatomical placode in mammals and birds, which gives rise to hair and feathers.

    37. skin developmental series (Fig. 1A) in crocodiles (Crocodylus niloticus), bearded dragon lizards (P. vitticeps), and corn snakes (Pantherophis guttatus)

      The authors took successive microscopic images of different body parts in both lizards and snakes, specifically focusing on the places that scales form.

    38. indicate that early scale morphogenesis in reptiles consists of regular dermoepidermal elevations that typically further develop into oriented asymmetrical scales with various levels of overlap, depending on the species and body area

      As was found in previous studies, scale development begins with the formation of risen areas on the skin. The symmetry and degree of overlap of the final scales depends on the species and location on the body.

    39. Extant

      Exists today.

    1. knockout

      A technique that allows a gene to be shut down and made nonfunctional. Researchers knock out genes to see what happens when they aren't active.

    1. warm mixed forest

      A temperate biome that is slightly warmer than the global average, marked by distinct seasons, sometimes with dry or rainy seasons. The forests are, as the name implies, of mixed composition. In this particular biome the predominant trees are broadleaf (ex: maple or oak) and conifers (ex: pine).

    2. temperate conifer forest

      A biome found in temperate regions with warm summers, cool winters, and enough precipitation to support coniferous trees ("evergreens").

  2. Dec 2017
    1. pleurodont

      Tooth fused to the inner edge of the jaw. They are loosely attached and can regenerate if lost.

    2. autopod

      Part of the limb farthest from the body, such as the hand or foot.

    3. similar phenotypes in other vertebrates because of impairments of the EDA receptor (EDAR; a member of the TNF family) (18) or its ligand EDA, indicating a conserved role of this pathway in reptiles as well

      Similar phenotypes are observed in other vertebrates (including mammals and birds) when the same pathway is interrupted. This suggests the pathway is conserved in reptiles, too.

    4. homozygous for a codominant mutation

      The scaleless bearded dragons have two copies of the same alleles (Sca). This gene is codominant, meaning both alleles are expressed even in heterozygotes.

    5. superposed

      Placed on top of one another.

    6. The most marked and derived macropatterning of skin in reptiles is observed in snakes

      Compared to other reptiles, snakes' scale development is unique.

    7. argued homologous to those characterized in chicken

      The authors found that the patterns between patterning of the tracts that give rise to chicken feathers and those that give rise to reptile scales could be homologous.

    8. spatiotemporal development is highly similar between the two species

      In Nile crocodiles and bearded dragons, scales developed from a similar anatomical placode and patterned similarly both spatially and temporally.

    9. feathers are organized into discrete tracts associated to different body areas

      In chickens, feathers develop in 10 distinct areas (tracts) which correspond to different parts of the body. These tracts develop at different rates and have their own patterning.

    10. Bmp4 is also shown for lizard. Red double-headed arrows indicate the body region processed for sectioning.

      The expression of Bmp4 in lizards strongly suggests a developmental evolutionary link among all reptiles, as well as birds and mammals.

    11. immunohistochemistry

      The use of antibodies to detect specific proteins in a tissue (using the principle that antibodies will selectively bind to certain antigens).

    12. proliferating cell nuclear antigen (PCNA) analyses indicate a reduced proliferation rate of the placode epidermal cells

      PCNA is a protein involved in cell division, so it is used as a marker to locate cells that are actively dividing.

      This analysis showed that the cells of the placode were reproducing very slowly.

    13. nested subpopulation

      A subset of a larger population.

    14. histological analyses

      Analysis of the structure of a tissue.

    15. This set of new results coherently and conclusively indicates that most skin appendages in amniotes are homologous

      The authors' results suggest that, because the anatomical placode is crucial to the development of skin appendages in mammals, birds, and reptiles, these appendages are homologous.

    16. wild-type

      The "typical" phenotype that is seen in nature.

    17. TNF

      The tumor necrosis factor (TNF) superfamily is a group of proteins involved in programmed cell death.

    18. codominant

      A type of dominance where two alleles of a gene in a heterozygote are fully expressed.

    19. that is, they are difficult to identify on any specific embryo because they establish multiple tracts whose developmental timing and locations vary across the body

      The authors believe that previous studies have overlooked anatomical placodes in reptiles because they are difficult to see during development.

    20. transitorily in time and nonconcurrently in space

      Placodes are very short-lived during development and do not form in the same place.

    21. associated with the presence of an anatomical placode presenting all the characteristics observed in avian and mammalian placodes

      The authors of this study provide evidence for the theory that hair, feathers, and scales are homologous structures.

    22. similarities in signaling are due to independent co-option of these molecular pathways

      Although some scientists think that the similarities in development of hair, feathers, and scales are evidence that they are homologous, others believe that they evolved independently.

      They propose that these pathways served a different function in the common ancestor of mammals, reptiles, and birds, and that they were only later co-opted for the development of hair, feathers, and scales.

    23. Several studies (9, 18–23) have shown that conserved signaling pathways, evidenced by the expression of the Sonic hedgehog (Shh), β-catenin (Ctnnb1), ectodysplasin A receptor (Edar), and/or bone morphogenetic protein (Bmp) genes, are involved in skin patterning and early morphogenesis of all amniote skin appendages

      Several signaling pathways involved in skin development and patterning are conserved across species. Different authors have reached different conclusions about whether these pathways are homologous or whether they evolved independently.

    24. dermoepidermal elevations

      Bumps in the layer that joins the epidermis (outer layer of the skin) and the dermis (middle layer of the skin).

    25. follicular organs

      Small, spherical groups of cells that contain a cavity from which hair, teeth, feathers, etc. can grow.

    26. scutate

      Covered by bony or horny plates or scales.

    27. avian

      Related to birds.

    28. homology

      Similarity of structure, physiology, or development of different species that is a result of a common evolutionary ancestor.

    29. discrete developmental units through reaction-diffusion

      Morphogens (chemicals which direct the development of different structures) interact to form gradients or concentrate in different parts of an organism. This leads to patterns in the placement and arrangement of appendages.

    30. lineage

      Refers to evolutionary lineage (species linked by a common ancestor).

    31. molecular markers

      Specific molecules that, when present, indicate the presence of a structure or a particular stage of development.

    32. columnar cells

      Cells shaped like columns.

    33. ectodysplasin A

      A protein involved in cell signaling between two layers of skin (ectoderm and mesoderm). It is especially important in embryo formation and promotes the formation of hair follicles, sweat glands, and teeth.

    34. histological

      Related to the study of tissues.

    35. co-option

      The process by which a structure or pathway that evolved for one function gains additional functions.

    36. common ancestry

      The idea that two species share an ancestor somewhere in their lineage. Common ancestry is visualized in a phylogenetic tree.

    1. F. A. Ran et al., Cell 154, 1380–1389 (2013).

      This study investigated offset nicking as a means of reducing off-target modifications. The authors found that by using Cas9 mutants with sgRNAs, a targeted double-stranded break can be performed. Off-target activity was reduced 50- to 1000-fold with this technique.

    2. K. T. Flaherty et al., N. Engl. J. Med. 363, 809–819 (2010).

      This study investigates the BRAF mutation in melanoma and the BRAF protein kinase inhibitor vemurafenib (PLX). The researchers found that PLX reduced tumor growth in cells that had a mutated BRAF gene.

    3. P. Mali et al., Science 339, 823–826 (2013).

      This study demonstrated that Cas9 is effective at introducing specific loss-of-function mutations in the genome and creating specific double-stranded breaks in DNA sequences.

    4. GeCKO can identify essential genes and that enrichment analysis of depleted sgRNAs can pinpoint key functional gene categories in negative selection screens.

      The significant decrease in the diversity of sgRNAs present at the end of the 14-day screen suggested that the GeCKO library was an effective way to identify survival genes.

      Because of this, the authors concluded that they could use this method to identify genes essential for cell function.

    5. when targeted to coding regions of genes can create frame shift insertion/deletion (indel) mutations that result in a loss-of-function allele

      Using a CRISPR RNA (crRNA) that is base-paired to a trans-activating crRNA (tracrRNA) will allow the scientists to direct the Cas9 to a specified area of the DNA and cause a double-strand break at the target location.

    6. The RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats)–associated nuclease Cas9 provides an effective means of introducing targeted loss-of-function mutations at specific sites in the genome

      The CRISPR-Cas9 system can be used to target and remove specific sequences from DNA. This is done by using a guide RNA that directs the CRISPR/Cas9 system to the correct location in the DNA sequence.

    7. predominant method for genome-wide loss-of-function screening

      RNAi is triggered by double stranded RNAs, which are processed into small interfering RNAs (siRNAs). siRNAs target complementary RNAs for destruction, which inhibits gene expression and allows for creation of knockouts.

      Human and Drosophila RNAi libraries have been made to map out the effectiveness of RNAi and the knockout ability in particular genes. Vector-based short hairpin RNAs are artificial molecules that can be used to selectively target genes. Libraries of these molecules can be used for genetic screens in both short-term and long-term assays.

      Although RNAi is a very effective tool, there are sometimes problems with completely turning off specific genes or targeting the correct gene.

    8. Human Genome Project

      The Human Genome Project was an effort to map and understand all of the genes that make up the human genome. Although it was announced complete in 2003, we still have much to learn about the genome.

    9. GeCKO

      Genome-scale CRISPR knockout.

  3. Nov 2017
  4. Oct 2017
    1. L. A. Gilbert et al., Cell 154, 442–451 (2013).

      This study investigated how different Cas9 mutants are more effective for different situations.

    2. S. R. Whittaker et al., Cancer Discovery 3, 350–362 (2013).

      This study investigated PLX drug resistance in A375 cells. The authors found that NF1 is the most common mutation leading to PLX resistance, a finding that is supported by the current study. The Raf inhibitor vemurafenib was used in both studies. The authors in this paper used a pooled RNAi screen that targeted 16500 genes to look at loss-of-function mutations leading to PLX resistance.

    3. A. L. Jackson et al., RNA 12, 1179–1187 (2006).

      This paper investigates some of the unintended consequences of RNAi, such as the lack of specificity. While one sequence may be the target, other sequences could also be silenced in this process. If a sequence shows some complementarity to the target sequence, it may be targeted, as well. The paper predicts that perfect specificity will be difficult to achieve with RNAi.

    4. J. E. Carette et al., Science 326, 1231–1235 (2009).

      This study is another example of a loss-of-function screen. The initial experiment found host factors that were necessary for influenza infection, such as genes important for the synthesis pathway of diphthamide and genes that are necessary for cytolethal distending toxin. The authors knocked these genes out to investigate their effects.

    5. M. Boutros et al., Heidelberg Fly Array Consortium, Science 303, 832–835 (2004).

      This study uses RNAi to screen for loss-of-function alleles. The initial experiment tested the function of 91% of the genome of Drosophila. Testing was done by using an RNAi of the known 19,470 genes involved in cell growth and viability.

    6. Whereas RNAi reduces protein expression by targeting RNA, GeCKO introduces loss-of-function mutations into genomic DNA

      For more on the differences between RNAi and CRISPR, see this article in Genetic Engineering and Biotechnology News:

      https://www.genengnews.com/gen-articles/advertorial-comparing-rnai-and-crispr-technology-for-loss-of-function-genetic-screens/5496

    7. which may be alleviated using an offset nicking approach

      Cas9 nickase creates a break in one strand of the double-stranded helix. These nicks are repaired with a very high fidelity.

      Using Cas9 with off-set sgRNA simultaneously nicks both strands, and may lead to a reduction of off-target modifications.

    8. Interestingly, although all five sgRNAs conferred resistance to PLX, only the best shRNA achieved sufficient knockdown to increase PLX resistance

      Not only did the authors find that GeCKO more consistently identified candidate genes, they found that the genes that were identified were better at providing PLX resistance.

    9. Western blot

      A technique used to determine which proteins are present in a sample.

    10. Lower P values for the GeCKO versus shRNA screen indicate better scoring consistency among sgRNAs

      The authors compared the top 100 hits in each screen and found that the sgRNA screen using GeCKO was more effective.

    11. we used several methods to evaluate the degree of consistency among the sgRNAs or shRNAs targeting the top candidate genes

      The authors compared GeCKO screening to a similar screen performed with shRNA.

    12. Our highest-ranking genes included previously reported candidates NF1 and MED12 (18, 19) and also several genes not previously implicated in PLX resistance, including neurofibromin 2 (NF2), Cullin 3 E3 ligase (CUL3), and members of the STAGA histone acetyltransferase complex (TADA1 and TADA2B)

      The RIGER algorithm identified NF1 (neurofibromin, a tumor suppressor protein that inhibits RAS), MED12 (helps regulate transcription of RNA polymerase II-dependent genes), NF2 (gene for merlin which is used to control cell shape, movement, and communication), CUL3 (plays a key role in the polyubiquitination and degradation of proteins as a component of the E3 ubiquitin ligase), TADA1 (subunit of STAGA a chromatin modifying complex of proteins), and TADA2B (promotes transcription by organizing HAT activity). All were implicated in PLX resistance.

    13. epigenetic

      Genetic changes that do not involve a change in DNA sequence and involve external or environmental factors.

    14. neurofibromatosis

      A genetic disease in which tumors develop on nerve tissue (including the brain, spinal cord, and nerves).

    15. These candidates yield new testable hypotheses regarding PLX resistance mechanisms

      The authors were able to identify new candidate genes for PLX resistance, and suggest that further research be done on how these genes confer this resistance.

    16. RNAi Gene Enrichment Ranking (RIGER) algorithm

      The authors used an algorithm to rank the genes enriched in their screen by how likely it is these genes contribute to PLX resistance.

    17. we found enrichment of multiple sgRNAs that target each gene after 14 days of PLX treatment (Fig. 3E), suggesting that loss of these particular genes contributes to PLX resistance.

      After 14 days, the authors saw a change in the distribution of sgRNAs in drug-resistant cells. From the new distribution of sgRNAs, they were able to identify genes that may contribute to PLX resistance.

    18. enrichment of a small group of cells that were rendered drug-resistant by Cas9:sgRNA-mediated modification.

      PLX exposure stops growth in cells with a specific BRAF mutation. Treating a group of cells with PLX will halt the growth of cells without resistance, but cells that are resistant to PLX will continue to grow. This allowed the researchers to isolate drug-resistant cells.

    19. we sought to identify gene knockouts that result in resistance to the BRAF protein kinase inhibitor vemurafenib (PLX) in melanoma

      To test the GeCKO library's effectiveness for positive selection (genetic variation that is beneficial to the cell), the authors tried to use it to identify which genes result in resistance to PLX.

      PLX is a BRAF enzyme inhibitor. BRAF is involved in the uncontrolled (cancerous) cell growth in melanoma.

    20. we conducted a negative selection screen by profiling the depletion of sgRNAs targeting essential survival genes

      To determine how effective the GeCKO library is at knocking out targeted genes, the authors first used a negative selection screen (which tests for deleterious effects).

      They infected cells with the library of sgRNAs. At the end of 14 days, they observed a reduction in the diversity of sgRNAs, as those sgRNAs that targeted genes necessary for survival were lost when cells died.

    21. we designed a single lentiviral vector to deliver Cas9, a sgRNA, and a puromycin selection marker into target cells (lentiCRISPR)

      For more on how lentiviral vectors aid in the use of CRISPR, see this article in Science

      http://science.sciencemag.org/content/341/6148/833.full

    22. endogenous

      Substances that naturally occur in a cell.

    23. We designed a library of sgRNAs targeting 5′ constitutive exons (Fig. 2A) of 18,080 genes in the human genome with an average coverage of 3 to 4 sgRNAs per gene (table S1), and each target site was selected to minimize off-target modification

      The authors produced a wide variety of sgRNAs that were used to find the 5’ end of constitutive exons, sequences that are constantly turned on and being used to create proteins.

    24. we tested the feasibility of conducting genome-scale CRISPR-Cas9 knockout (GeCKO) screening with a pooled lentiCRISPR library

      See AddGene for more information about designing sgRNAs and genome-scale sgRNA libraries (including the GeCKO library):

      https://www.addgene.org/crispr/guide/

    25. lentiCRISPRs abolished EGFP fluorescence in 93 ± 8% (mean ± SD) of cells after 11 days

      The efficacy of gene knockout by lentiCRISPR transduction was high. The sgRNAs eliminated 93+/- 8% of EGFP fluorescence in the experiment.

      Further, lentiCRISPR transduction was more effective than transduction of cells with lentiviral vectors expressing EGFP-targeting shRNA, which produced an incomplete knockdown of EGFP.

    26. transduction

      The process of genetic material being incorporated into a cell through the use of a virus.

    27. they can be easily titrated to control transgene copy number and are stably maintained as genomic integrants during subsequent cell replication

      Lentiviral vectors are commonly used for gene insertion because of their stability. It is easy to control the number of vectors, and once integrated into the gene the inserted DNA segment is likely to stay there.

    28. Lentiviral vectors

      Lentiviruses are a group of viruses that incorporate their own RNA into the DNA of the host cell they infect. They can infect both dividing and nondividing cells.

      Modified lentiviruses that carry experimental RNA molecules are called "lentiviral vectors." Researchers can use them to insert, modify, or delete genetic material in an organism.

    29. potential of Cas9 for pooled genome-scale functional screening

      As noted above, genome-wide screening has been successfully performed with RNAi. Here, the authors wanted to know if CRISPR could offer a more accurate and precise way to screen genomes.

    30. oligonucleotide

      Short DNA or RNA molecules that form a chain of approximately 20 nucleotides.

    31. single-guide RNA (sgRNA)

      A synthetic RNA that guides Cas9 to a specific spot on a DNA molecule.

    32. RNA interference (RNAi)

      A process that uses RNA molecules to inhibit genes, usually by destroying specific messenger RNA.

    33. therapeutic RAF inhibitor

      A type of drug used to treat cancers. These drugs help regulate genes that are disrupted by cancer.

    34. melanoma

      A cancer in the pigment-producing cells of the skin.

    35. library

      Compilation of DNA fragments which can be used to determine which genes in a genome to alter and knock out.

    36. the promise of genome-scale screening with Cas9

      For more on possible future uses of CRISPR-Cas9, see this article in Science Daily:

      https://www.sciencedaily.com/releases/2016/08/160811142643.htm

    37. interrogate

      Investigate.

    38. CRISPR

      A technique that allows for targeted changes in the genome by cutting, replacing, and adding gene sequences.

    1. monogenic neurological disorders

      Disorders caused by a single gene.

    2. homozygous deletions

      Deletions in which the genetic information is missing on both chromosomes.

    1. Scaleless dragons show an irregular skin surface with the initiation of some dermoepidermal undulations of the skin (Fig. 3G), indicating that this phenomenon does not fully require the presence of anatomical placodes.

      Mutant dragons displayed evidence of scale patterns, despite the absence of fully formed scales. This shows that there are factors other than the anatomical placode at play in scale formation.

    2. morphogenesis

      The formation of an organism's structures and features.

    3. homologous

      Homologous properties in biology have the same evolutionary origins, but not necessarily the same function. A common example is the human arm and a bat's wing.

    4. dermoepidermal elevations

      Bumps found in the area of tissue that joins the epidermal (outer) layer and the dermal (middle) layers of the skin.

    1. narrow-and-deep approach

      This refers to results of studies that go into detail on a specific area, without covering a wide range of different topics.

    2. (such as experience and expertise)

      The expertise of researchers conducting the replication attempts has been the topic of much debate.

      In a recent study, Protzko and Schooler have conducted a study on the question whether researchers' "caliber" influences their success in reproducing studies.

      Read more in PsyArXiv Preprints:

      https://osf.io/preprints/psyarxiv/4vzfs/?t=1&cn=ZmxleGlibGVfcmVjc18y&iid=c229da44ef46429fb9c1547524d11052&uid=1158994320&nid=244+276893704

    3. Also, the replication “succeeds” when the result is near zero but not estimated with sufficiently high precision to be distinguished from the original effect size.

      Here, the authors describe a problem of judging the replication success by evaluating the replication effect against the original effect size. When the replication effects size is near zero, it could be possible that the data shows no effect, and therefore we would find an unsuccessful replication attempt.

      However, the estimation of the effect size could be imprecise. This means that there could be a lot of “noise” in the data, from random or systematic errors in the measurement. If there was a lot of noise in the data, it could distort our impression of whether the effect is really zero or not.

      We might conclude that a replication with an effect size close to zero was sufficiently different from zero and thus successful, although the effect was really just produced by noise in the data, and the true effect is zero, meaning that the replication could be falsely taken as a success.

    4. A key weakness of this method is that it treats the 0.05 threshold as a bright-line criterion between replication success and failure (28)

      Braver, Thoemmes, and Rosenthal (28) argue that judging the success of a replication only by whether it shows a significant effect (in the current study, at the 0.05 threshold) would be inappropriate.

      They argue that replication success depends a lot on the statistical power and therefore on the sample size used in the replication study. The replication study must have sufficiently many subjects so that it is probable enough that the effect in question, should it really exist in the population, can be found in this sample. If a replication study had low power, for example because the size of the original effect was overestimated and the replication sample size was consequently too small, this makes it less likely that the replication attempt will be successful and show a result that is statistically significant at the 0.05 threshold.

      For each individual replication study, the replication success therefore depends on the sample size. If you assess several replication attempts individually, the replication success rate could therefore be distorted to underestimate how reproducible an effect really is.

      To circumvent this problem, the authors suggest using a different technique than counting if individual replications were significant at the 0.05 threshold. They analysis is called “continuously cumulating meta-analysis”. The data of several replication attempts are combined, so that conclusions on whether the data of all the replication attempts supports the effect of interest.

      After a new replication attempt is conducted, its data is added to the pool of data from previous replication attempts. This data is then taken together, and on the combined data, a test is run to estimate the effect of interest.

    5. Results

      The authors used 5 measures for replication success to check to what extent the 100 original studies could be successfully replicated.

    6. The overall replication evidence is summarized in Table 1 

      The authors wanted to know if successfully replicable studies differed from studies that could not be replicated in a systematic way. As the criterion for replication success, they used their first approach (significance testing). They found that studies from social psychology and studies from social psychology journals were less likely to replicate than those stemming from cognitive psychology and cognitive psychology journals. Moreover, studies were more likely to replicate if the original study reported a lower p-value and a larger effect size, and if the original finding was subjectively judged to be less surprising. However, successfully replicated studies were not judged to be more important for the field or conducted by more experienced research teams.

    7. and 25%

      Prinz and colleagues (11) comment on their experience as employees of a pharmaceutical company, which relies on preclinical research to decide whether to invest into the exploration and development of new drugs. Because companies find many preclinical research findings unreliable, they now often conduct their own research to reproduce the original findings before they decide to move on and invest large sums of money into the actual drug development.

      Only in about 20% to 25% of the cases did the company scientists report finding results of the reproduction that were in line with the originally reported findings.

    8. in only 11

      Begley and Ellis (10) are cancer researchers, who propose ways for research methods, publication practices and incentives for researchers to change so that research would yield more reliable results, such as more effective drugs and treatments. They argue that often new drugs and treatments enter clinical trials, which test their effectiveness to treat cancer in humans, before they reach sufficient standards in preclinical testing, leading to non-reproducible findings. To achieve more reliable preclinical results, they argue that more focus should be placed on reproducing promising findings in the preclinical phase.

    9. insufficient specification of the conditions necessary or sufficient to obtain the results (12–23)

      Making an experimental setup transparent and reproducible can be quite difficult, because undetected but theoretically relevant variations to the study protocol could produce different results. However, there are some new ideas about how such transparency could be achieved.

      Read more in The New Yorker: http://www.newyorker.com/tech/elements/how-methods-videos-are-making-science-smarter

    10. psychology or other disciplines

      Reproducibility has become the focus of attention in many areas of science. So far, you have read about examples from cancer research and biology, as well as pharmaceutical research. Another scientific discipline that has recently begun addressing the topic of reproducibility is the field of economics. In their approach to evaluating the reproducibility of studies in experimental economics, Camerer and colleagues have also used a somewhat different methodology.

      Read more in Science:

      http://science.sciencemag.org/content/351/6280/1433.full.pdf+html

    11. Moreover, correlational evidence is consistent with the conclusion that variation in the strength of initial evidence (such as original P value) was more predictive of replication success than was variation in the characteristics of the teams conducting the research (such as experience and expertise).

      From this study, we can conclude that the efforts made to reduce the influence of the researchers involved in the replication studies on replication success seemed merited. Importantly, replication success was less influenced by the characteristics of the replication team than by how strong the results of the original study were. Stronger evidence for the effect the original study investigated was related to a successful replication.

    12. The disadvantage of the descriptive comparison of effect sizes is that it does not provide information about the precision of either estimate or resolution of the cumulative evidence for the effect.

      For the fourth measure, the authors combined the original and replication effect sizes and calculated a joint estimation of the effects. They wanted to see how many of the studies that could be analyzed this way would show an effect that was significantly different from zero if the evidence from the original study and that of the replication study was combined.

      Results showed that 68% of the studies analyzed this way indicated that an effect existed. In the remaining 32% of the studies, the effect found in the original study, when combined with the data from the replication study, could no longer be detected.

    13. A complementary method for evaluating replication is to test whether the original effect size is within the 95% CI of the effect size estimate from the replication.

      A second way to evaluate the replication success was to compare the sizes of the effects of the original studies and those of the replication studies using confidence intervals (CIs). They checked if the effects size of the original study was a value that was also among the range of values revealed as good estimates of the true effect size in reality, which was calculated from the size of the effect in the replication study.

      Using this measure, they found that slightly fewer than half the replications were successful.

    14. A straightforward method for evaluating replication is to test whether the replication shows a statistically significant effect (P < 0.05) with the same direction as the original study.

      The authors first checked how many replications "worked" by analyzing how many replication studies showed a significant effect with the same direction (positive or negative) as the original studies.

      Of the 100 original studies, 97 showed a significant effect. Based on their ability to replicate the original studies, the authors expected around 89 successful replications. However, results showed that only 35 studies were successfully replicated.

    15. We correlated the five indicators evaluating reproducibility with six indicators of the original study (original P value, original effect size, original sample size, importance of the effect, surprising effect, and experience and expertise of original team) and seven indicators of the replication study (replication P value, replication effect size, replication power based on original effect size, replication sample size, challenge of conducting replication, experience and expertise of replication team, and self-assessed quality of replication)

      Last, the authors wanted to know if successfully reproducible studies differed from studies that could not be replicated in a systematic way.

      For this, they checked if a number of differences in the original studies, such as the size of the effect originally reported, was systematically related to successfully replicated studies.

      They also checked if a number of differences in the replication studies themselves, such as the size of the effect of the replication study, related systematically to successful replications.

    16. We conducted fixed-effect meta-analyses using the R package metafor (27) on Fisher-transformed correlations for all study-pairs in subset MA and on study-pairs with the odds ratio as the dependent variable.

      The authors combined the results of each original and replication study to determine if the cumulative joint effect size was significantly different from zero. If the overall effect was significantly different from zero, this could be treated as an indication that the effect exists in reality, and that the original or replication did not erroneously pick up on an effect that did not actually exist.

    17. tested the hypothesis that this proportion is 0.5

      The authors hypothesized that in half the replication studies, the effect would be stronger in the original than the replication.

      The reason for choosing this null hypothesis here is that this is the expectation we would have if chance alone determined the effect sizes. There are two likely outcomes for each replication study: (1) that its effect size is bigger than that of the original study, and (2) that its effect size is smaller than that of the original study.

      If chance alone determined the effect size of the replication, we would see each possible outcome realized in 50% of the cases. If the proportion of replication effects that are bigger than the effect of the original study was significantly different from 50%, it could be concluded that this difference was not random.

    18. null hypothesis

      The null hypothesis is the assumption that a certain effect does not exist in reality, and that any observations of this effect in data is due to unsystematic error.

    19. predictors

      Predictors are variables that the researchers identified as potentially correlated with reproducibility.

    20. Each replication team conducted the study, analyzed their data, wrote their summary report, and completed a checklist of requirements for sharing the materials and data.

      After completing their replication attempt, independent reviewers checked that each team's procedure was well documented, that it followed the initial replication protocol, and that the statistical analysis on the effects selected for replication were correct.

      Then, all the data were compiled to conduct analyses not only on the individual studies, but about all replication attempts made. The authors wanted to know if studies that replicated and those that did not replicate would be different.

      For instance, they investigated if studies that replicated would be more likely to come from one journal than another, or if studies that did not replicate would be more likely to have a level of statistical significance than studies which could be replicated.

    21. correlation coefficient (r)

      A correlation coefficient describes the statistical relationship between two variables. It shows both the direction (positive coefficient: as A increases, B increases as well; negative coefficient: as A increases, B decreases), and the strength of the relationship (coefficient close to zero: weak relationship; coefficient close to +/- 1: strong relationship).

      For example, there might be a positive correlation between years of attendance to school and general knowledge: the longer people have attended school, the more knowledge they acquire. On the other hand, there could be a negative correlation between hours spent watching TV and enjoyment of outdoor activities: the more time people spend watching TV, the less they might enjoy going for a hike.

      Importantly, correlation coefficients do not tell us anything about causation.

    22. likely that more than half of research results are false and therefore irreproducible (9)

      Ioannidis conducted computer simulations to show that for most studies, it is more likely for a finding to be a false positive than a true identification of an effect.

      Among the factors that make it more likely for research findings to be false are small sample size or the underlying effect, and when designs, definitions, and analyses are more flexible rather than rigorously objective.

    23. sufficient

      Sufficient conditions are one set of circumstances under which a specific effect can be found, but there could be many other circumstances under which the effect would occur.

      For example, if a person asks you for money and you give it to them, asking for money is a sufficient condition. It's enough to make you give the person money. But there are other circumstances in which you would have done the same. For example, you may have given them money in exchange for a bouquet of flowers.

    24. necessary

      Necessary conditions are the circumstance that must be met in order to find a specific effect. If these conditions are not met, the effect cannot be found.

      For example, to find the effect that prosocial people are more likely than selfish people to give change to someone asking for money, a necessary condition would be studying human subjects, not penguins.

    25. effect sizes

      The strength of an effect.

    26. statistically significant

      Results are referred to as statistically significant when we find the result convincing because it is extremely unlikely that the observed effects are due to random chance.

    27. Reproducibility

      Reproducibility is a characteristic of a scientific study, stating that it can be run and run repeatedly, and will in its repetitions yield the same result.

      If an experiment has been reproduced successfully, it has been conducted more than once with similar results each time. Subsequent studies are called reproducing studies, replication studies, or replications.

    1. T. F. Goreau, Ecology 40, 67 (1959).

      Goreau describes the species of corals found in Jamaica's reefs and the patterns of their distributions. He notes that the northern and southern coasts look different from each other and hypothesizes that these differences are driven by differences in storm frequencies.

    2. fecund

      Fecundity refers to the ability of an organism to produce offspring. If an organism is more fecund, this means it produces more offspring

    3. brittle stars

      Sea stars from the class Ophiuroidea. Brittle stars are named so for their fragile, thin arms.

    4. Diadema antillarum

      Diadema antillarum is commonly known as the long-spined sea urchin. Before populations of D. antillarum died off in 1983 in massive numbers, they were a common sight in Caribbean reefs and played an important role in controlling the growth of macroalgae.

    5. arborescent gorgonians

      Gorgonians are part of a group of corals often called "soft corals" due to their lack of a rigid calcified skeleton. Arborescent means "treelike," referring to gorgonians that specifically have upright treelike forms.

    6. Agraria spp.

      Agaricia is a genus of corals that form flat leaf-like or plate-like structures. The "spp." means "multiple species of." So Agaricia spp. means "species of the genus Agaricia."

    7. Acropora palmata

      Acropora palmata is commonly known as Elkhorn coral for its antlerlike appearance. As a result of its complex 3D shape, A. palmata adds significant structure to coral reefs and forms an important habitat for many other marine organisms.

    8. In the last half-century, while Port Royal on the south coast experienced 11 hurricanes, Discovery Bay on the north has seen only four; the last severe hurricane was in 1917 (12).

      Since this study was published, hurricanes have been increasing in both frequency and intensity in the North Atlantic. We may begin to see shifts in coral reef communities toward something more like Port Royal.

      Read more from the U.S. Global Change Research Program:

      http://nca2014.globalchange.gov/report/our-changing-climate/changes-hurricanes

    1. microstimulation

      A technique that stimulates a small population of neurons by passing an electrical current through a nearby microelectrode.

    2. Deep-layer trunk cortex

      The cortex is divided into layers from the surface of the brain inward. The deep-layer levels of the trunk cortex are closer the the center of the brain.

      Diagram of the cortical layers of a human brain: https://www.ncbi.nlm.nih.gov/books/NBK10870/figure/A1798/

    3. somatosensory cortex

      The part of the brain that detects touch happening in certain places in the body.

      The trunk region of the somatosensory cortex specifically activates when the trunk, or the main body, of the rat is stimulated.

      Diagram of the human somatosensory cortex: https://www.ncbi.nlm.nih.gov/books/NBK11153/figure/A641/?report=objectonly

    1. RT-PCR

      Reverse transcriptase polymerase chain reaction is a variation of polymerase chain reaction (PCR). RT-PCR begins with an "extra" step in which RNA is reverse transcribed into its DNA complement and then amplified using traditional PCR.

      It is commonly used to analyze the expression patterns of infections and diseases.

  5. Sep 2017
    1. natural experiment

      When natural events (i.e. fires, hurricanes, or other disturbances) happen to only some areas, scientists can study the effects by comparing affected and unaffected sites.

      This is a natural experiment.

    2. While hurricanes can cause violent disturbance to coral reefs with extreme short- (5) and long-term (3, 4, 6-9) effects, very little is known of their' immediate consequences for previously investigated populations (10)

      Various studies describe the aftermath of tropical cyclones on coral reefs. Effects of storms seem to vary depending on the structure, form, and composition of the affected reef.

      None of the studies cited had intimate knowledge of the impacted reefs immediately before the disturbance.

    3. community composition

      A community is a group of interacting organisms that live in the same location. Community composition is the makeup of a community.

    4. The relative importance of environmental processes that affect the distribution of organisms varies with the intensity and frequency of the processes

      Disturbances in the environment, such as storms and fires, can affect the state of an ecological community.

      For example, traditional ecological knowledge dictates that when disturbances are frequent or intense, all species in the affected area may become locally extinct. But when disturbances are rare, the community may become dominated by only the most competitive species.

      Counterintuitively, an intermediate level of disturbance may maintain the highest level of biodiversity.

    5. Coral reefs

      Corals are a group of colonial marine animals that form hard calcium skeletons. They have stinging cells that can be used to catch and kill small prey, but they also often carry symbiotic algae inside that they can use to convert sunlight into cellular energy.

      When corals live together in large groups, they can form a buildup of sediment and minerals in the ocean called a reef. The complex structures of reefs create habitats for many other ocean animals.

    1. gene regulatory networks

      https://www.sciencedaily.com/releases/2016/07/160728110501.htm

      The Institute for Basic Science research team explained the use of a technique, mTAIL-seq, to study the regulation of translation through the presence and length of the poly-A tail.

      A basic background of the process of transcribing DNA into RNA and translating RNA into proteins is given in the article to show the importance of each step of the central dogma.

      The IBS research team was able to use their technique to show that the regulation of the messenger RNAs controls what proteins are being expressed in the cell controlling the phenotype of the individual: hair color, height, etc.

    2. with MS2 to image single mRNA translation kinetics in live cells

      http://science.sciencemag.org/content/352/6292/1430

      Bin Wu lead a group of researchers in developing molecular techniques which would allow translation elongation and initiation rates to be observed and quantified. Form Wu’s research, the SunTag epitope was developed and combined with MS2 tagging, which was the first experiment that allowed movement of mRNA, translation, and protein migration to be observed in a living cell.

      Wu’s research on translation kinetics helped lay the foundation for Morisaki’s research and the development of nascent chain tracking.

    3. SunTag

      System of tagging RNA so the movement of mRNA in the cell can be seen.

    4. This work is similar to a companion manuscript by Wu et al.

      This paper is in the same issue of Science and describes a different way to visualize translation in the cell using fluorescent markers.

    5. we found the complexes to be roughly twice the size of a single polysome

      The biggest molecule, in this experiment KDM5B, took the longest to translate.

    6. there was little interaction between the two, providing direct evidence that the vast majority of KDM5B polysomes act independently of one another. However, a small fraction (~5%) of KDM5B polysomes formed complexes that co-moved for hundreds of seconds

      Most polysomes translated the FLAG tagged and HA tagged proteins independently; however, a few interacted with one another, suggesting that translation of some proteins might be co-regulated.

    7. In parallel, we engineered a new KDM5B construct with a 10X HA-tag SM (HA-KDM5B) (5) to complement FLAG-KDM5B (formerly referred to as SM-KDM5B), as shown in Fig. 4A. As a first application of this technology, we wanted to test if polysomes interact with each other to form higher-order structures that can translate two distinct mRNAs at the same time.

      To determine whether multiple polysomes can interact with one another to translate multiple mRNAs, the authors transfected two KDM5B genes into the cell, one containing the HA SM tag and one containing the FLAG SM tag. These two tags can be detected by different antibodies bound to different color fluorochromes.

    8. HA

      A protein used by influenza virus to enter cells and infect them. Here it is used to as a tag to label proteins as they are translated so that translation and protein movement can be seen.

    9. yielding a single consistent elongation rate of 10 ± 2.3 aa/sec, fairly close to what has been measured using genome-wide ribosomal profiling (5.6 aa/sec)

      The translational elongation rate was consistent for each of the translated proteins and was between 8-12 amino acids per second.

    10. Importantly, for all constructs, the correlation vanished at times greater than the dwell time. This implies initiation is random, so there is no memory between initiation events, similar to what was observed for transcriptional initiation (12) and in contrast to bursting

      Like the process of transcription, the start of the translation process is random and there is no memory of previous initiations.

    11. cycloheximide

      An organic compound that interferes with protein synthesis

      https://upload.wikimedia.org/wikipedia/commons/4/48/Cycloheximide.png

    12. photobleaching

      Fading of the fluorophore to stop the fluorescence of the protein

    13. To measure the lifetime of Fab binding, we performed fluorescence recovery

      Photobleaching or FRAP uses laser light to quench the fluorescence being emitted by the fluorescent tag. The fluorescence can be recovered by Fab antibodies leaving the translated proteins and being replaced by new antibodies. Thus, this experiment can determine how length of time that the Fab antibodies can bind to the newly translated proteins. These experiments were used to gather baseline data to determine the rate of transcriptional elongation.

    14. To measure how quickly Fab bind polysomes, we microinjected them into cells transfected 6 hours earlier with our KDM5B construct and pre-loaded with MCP.

      This experiment measured how quickly the Fab antibodies bound to the polysomes by injecting the antibodies into cells containing the KDM5B gene and the MCP fluorescent marker.

    15. fluorescence correlation spectroscopy (FCS)

      Procedure that compares the fluorescent intensity between different molecules

    16. This suggests that the polysomes we imaged are organized in a more globular shape rather than an elongated shape, consistent with recent atomic force microscopy images

      Polysomes used for translation are constructed like a big ball rather than an elongated structure.

    17. Gaussian fitting

      A statistical method used to show the normal distribution of data

    18. diffraction limited spots

      A restricted area that is seen through the lens of a microscope

    19. 3′ UTR

      Untranslated region of the mRNA at the 3’ end