Interplay Between a Protein and a Ligand Assignment | Online Assignment

Description

For this assignment you are asked to investigate, at the molecular level, the interplay between a protein and a ligand with which it interacts. You are free to investigate a protein- ligand interaction of your own choosing and that you find interesting (many examples are provided below to help you make a choice). You will plan your literature research, gather information about the protein, its ligand and the function induced by this interaction, then use this information to write a report on the major features of the protein-ligand complexBCH2IBM – Integrated Research Assignment on Protein-Ligand Interactions
List of possible topics/protein-ligand pairs
AMA1 – RON2 (malarial proteins)
Angiotensin Converting Enzyme – Angiotensin I
Antibody – Antigen (HER2 Human Epidermal Growth Factor Receptor 2 (Neu) – Herceptin mAb)
Avidin – Biotin
Calmodulin – Calcium
DHPR (Dihydropyridine Receptor) – RYR (Ryanodine Receptor)
Dihydrofolate Reductase (DHFR) – DHF, THF
DNA Helicase – DNA
Erythropoietin (EPO) Receptor – Erythropoietin (EPO)
F1F0 ATP Synthase – ATP, ADP & Pi
Ferritin – Iron
Galactosidase – Lactose
GPCR (Dopamine Receptor – Dopamine)
Haemoglobin – Haem
Histone H1 – DNA
Histone Chaperones (Chaperone NAP1 – Histones)
HIV Protease – HIV Protease Inhibitor
HSP70 Chaperone – Proteins
Insulin-like Growth Factor 1 Receptor (IGF1 R) – IGF1
Insulin Receptor – Insulin
Intrinsic Factor (protein) – Vitamin B12
Lac Repressor – Lactose
Lectin – Sugar (Concanavalin A – Mannose/Glucose)
Leptin Receptor – Leptin hormone
Ligand-Gated Ion Channel/LGIC (Nicotinic acetylcholine receptor/nAChR – AcCh)
Mad Cow Disease PrPsc-PrPc (protein – prions)
Myosin – Actin
Na/K ATPase – Na, K
Neuroglobin – Nitric Oxide
NMDAR (N-methyl-D-aspartate receptor, a glutamate-type R) – NMDA
Phytases – phytate
Protein Kinase A – Cyclic AMP
PTEN (Phosphatase & Tensin Homolog/phosphatidylinositol-3,4,5-triP 3-P’ase) – PIP3
RNA Polymerase – Sigma subunit
Splicesome – RNA
TATA Box Binding Protein (TBP) – DNA
TDP-43 – mRNA (relevant to ALS disease)
Thrombin – Fibrinogen
Thyroid Hormone Receptor – Thyroid Hormone
Tissue Transglutaminase 2 – Ca2+ ions
Topoisomerase – DNA
Transferrin – Iron
Transferrin Receptor (TFR) – HFE1 Protein
Voltage-Gated Na Channel – Tetrodotoxin
Winter Flounder Anti-Freeze Proteins – Water/IceRESEARCH ASSIGNMENT
One of the major tasks of any scientist is to present and explain their work to others. Scientific research and the related fields of medicine, nutrition, agriculture and many others rely absolutely on sharing new discoveries and insights. Scientific research is reported in specialist journals and at scientific meetings where other scientists gather to hear the latest developments. New ideas and new explanations grow out of the work of many individual scientists who discuss their work together.
Background
Proteins are the most prevalent macromolecule in biological systems, and they participate in the majority of cellular functions. They are immensely diverse, varying significantly in size, structure and function. Proteins are involved in a multiplicity of processes including speeding up chemical reactions (i.e. the catalysis of essentially all reactions in biosynthesis and metabolism by enzymes), cell signaling (hormones and receptors), cellular transport (haemoglobin, neurotransmitter and ion transporters), cell structure (actin, tubulin, and keratin), motor function (myosin, dynein and kinesin) and defense mechanisms (antibodies and coagulation cascade proteins).
The function of proteins usually requires some form of interaction with another molecule. The molecule or substance which is bound to the protein to form a functional complex is referred to as a ‘ligand’. Many proteins interact with multiple ligands that can range from ions or other small molecules to other macromolecules, including other proteins and sometimes other copies of the same protein.
The task
For this assignment you are asked to investigate, at the molecular level, the interplay between a protein and a ligand with which it interacts. You are free to investigate a protein-ligand interaction of your own choosing and that you find interesting (many examples are provided below to help you make a choice).
You will plan your literature research, gather information about the protein, its ligand and the function induced by this interaction, then use this information to write a report on the major features of the protein-ligand complex. You should work independently on this exercise, which is considered as part of the theory component of BCH2IBM and which will contribute 10% to your final mark for the subject.
These notes provide information about:
(i) choosing a suitable topic (i.e. a protein-ligand interaction that meets our requirements)
(ii) selecting appropriate sources of information for your research;
(iii) ensuring that you acknowledge your information sources (to avoid plagiarism);
(iv) ensuring that you acknowledge your resources in the correct manner (by following the appropriate referencing conventions);
(v) blending the information to provide a well-structured, smooth-flowing, informative, cohesive and easy to read description of your chosen protein-ligand interaction;
(vi) how to use ‘Turnitin’ text-matching software to avoid inadvertent plagiarism and to improve the scientific integrity of your writing.
Objectives
The major aim of this task is to provide an exercise that will simultaneously refine your scientific writing skills and improve your understanding of the relationship between the structures and the biological functions of proteins.
• To develop research skills: to gather and use appropriate information from specialist texts, and primary and secondary literature sources;
• To develop scientific writing skills: to use appropriate vocabulary, select relevant information, write a comprehensive report for a general scientific audience;
• To develop correct referencing skills: in-text citations, reference list using a standardized format;
• To develop an understanding of protein interactions and how the complexes formed affect molecular function.
Choosing a protein – ligand interaction to work on:
Here are just a few suggested protein/ligand interactions you may choose for your assignment, together with a reference for each to help you get started on your research:
Protein
Ligand
A reference to get you started…
ATP synthase (FoF1ATPase)
ATP
Capaldi, R.A. & Aggeler, R. (2002) Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor. Trends Biochem Sci. 27(3), 154-60.
G protein-coupled receptors
G proteins
Oldham, W.M. & Hamm, H.E. (2007) How do receptors activate G proteins? Adv Protein Chem 74, 67-93.
Hsp70
Proteins
Goloubinoff, P. & De Los Rios, P. (2007) The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem Sci 32(8), 372-80.
Ferritin
Iron
Koorts, A.M. & Viljoen, M. (2007) Ferritin and ferritin isoforms I: Structure-function relationships, synthesis, degradation and secretion. Arch Physiol Biochem 113(1): 30-54.
Antibody/Immunoglobulin
Antigen
Davies, D.R., Padlan, E.A. & Sheriff, S. (1990) Antibody-antigen complexes. Annu Rev Biochem 59, 439-473.
Myosin
Actin
Geeves, M.A. & Holmes, K.C. (1999) Structural mechanism of muscle contraction. Annu Rev Biochem 68, 687-728.
Histone
Histone chaperones
Eg. NAP1, CAF-1, Spt6, FKPB, JDP2
Eitoku M., Sato L., Senda T. and Horikoshi M. (2008) Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly. Cell Mol Life Sci 65, 414 – 444.
RNA polymerase
σ (sigma) subunit
Murakami, K.S. & Darst, S.A. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13(1) 31-9.
Opsin/Rhodopsin
Retinal
Palczewski, K. (2006) G protein-coupled receptor rhodopsin. Annu Rev Biochem 75, 743-67.
Spliceosome: this is a complex of several U-rich small nuclear (sn)RNAs and many proteins that interact for the complex to exert its function.
Will, C.L. & Lührmann, R. (2006) Spliceosome structure and function. In RF Gesteland, TR Cech, JF Atkin, (eds) The RNA World, Third Edition, New York: Cold Spring Harbor Laboratory Press), pp. 369–400.
Lectin
Sugar
Van Damme, E.J.M., Peumans, W.J., Barre, A. & Rougé, P. (1998) Plant Lectins: A Composite of Several Distinct Families of Structurally and Evolutionary Related Proteins with Diverse Biological Roles, Critical Reviews in Plant Sciences 17(6), 575-692.
Na, K-ATPase
Na+, K+, ATP
Jorgensen, P.L., Hakansson, K.O. & Karlish, S.J.D. (2003) Structure and Mechanism of Na,K-ATPASE: Functional Sites and Their
Interactions. Annu. Rev. Physiol. 65,817–49.
On the LMS there is a more comprehensive listing of possible assignment topics. If you would like to research a particular protein-ligand interaction that is not on either of these two lists provided, contact the subject coordinator.
Resources
Journal articles and reviews
To research more advanced topics in later years of study, primary research articles provide the most appropriate source of information, however for this task at second year university level you may find that review articles offer more relevant information in a condensed and concise form. Try using an abstracting database (e.g. PubMed, Current Contents or Medline) to look for articles and reviews on the protein-ligand interaction that you have chosen.
Textbooks
You may find useful information about many of the suggested protein-ligand interaction topics in general introductory Biochemistry and Molecular Biology textbooks, for example:
(i) Nelson D.L. & Cox M.M. (2012) Lehninger: Principles of Biochemistry, 6th edn, W.H.Freeman and Co., New York [alternatively 5th edition (2008), 4th edition (2004)]
(ii) Lodish H., Berk A., Kaiser C.A., Krieger M., Scott M.P., Bretscher A., Ploegh H. and Matsudaira P. (2008) Molecular Cell Biology, 6th edn., W.H.Freeman and Co., New York
Websites
The web is a huge resource of information, some reliable and some not. Apart from websites run by the publishers of some scientific journals, very few web sites are peer-reviewed or checked for accuracy in the way that most printed scientific material is. For this reason, web sites will not be considered as acceptable ‘references’ for the purposes of this assignment.
You may use a web site to source a diagram if you wish, but keep in mind that this task is primarily an exercise in scientific writing, so a diagram is unlikely to gain you many marks and it should only be included if it really helps to explain the interaction that you are describing. If you include a web-sourced diagram, remember that it must be duly acknowledged (just as you would credit information from a printed journal article).
Despite not being able to use a web site as a direct reference in this assignment, be aware that the web can be an excellent starting point to find information about your topic. For example, although Wikipedia obviously cannot be used directly as a reference, it can lead you to primary research articles which can be cited directly in your assignment.
Format and assessment of your report:
Your written report should be presented in essay form using complete grammatically-correct sentences with correct punctuation and spelling. You should use paragraphs and/or headings to organize and add structure to the material presented.
The report should be 500 words in length (not counting in-text citations, diagrams, figure legends and your reference list) however a ±10% margin will be allowed on the word count.
Each piece of information that you present must be accompanied by an in-text citation to acknowledge its original source (unless the information is your own opinion). It is expected that you already understand how to correctly cite information sources, but some notes on correct referencing are provided later in this section.
Your report should discuss the following points, but they do not necessarily need to be presented point-by-point as listed below, in fact we want you to write fluently and avoid presenting what simply looks like a list of answers to a series of questions.
Points 1 – 5 are compulsory – your report must cover these aspects of your chosen interaction.
You should also cover at least one other feature of your chosen interaction – it’s up to you to decide which extra point(s) to cover, as some may be more suitable or more interesting than others depending on which topic you have chosen. Points 6 – 8 below are merely suggestions:
1. What are the components of the protein and ligand pair that you have chosen?
2. What is the primary biological function resulting from the interaction between this pair of molecules? What purpose does the interaction serve?
3. What major cellular activities (metabolic pathways/processes) require this interaction?
4. How do the protein and ligand interact at the molecular level? What are the structural requirements for this interaction to occur? What triggers the interaction?
5. Where does this interaction occur (tissue/organ, cell, subcellular organelle)?
—————————————————————————————————
6. (example) What happens if the interaction does not occur at all, or if it functions incorrectly, in nature?
7. (example) Can we artificially manipulate the interaction? If so, to what purpose?
8. (example) Is there any variation in the interaction in different cells or organisms?
References:
Use the Harvard referencing style, i.e. author-date format in-text citations (eg. ‘Jones and Smith, 1998’) followed by a reference list at the end of your report showing all of the references that you have cited in your report, presented in alphabetical order based on first-author surname. Information about referencing conventions is provided later in this section. Note that you should NOT use the numerical ‘Vancouver’ referencing style for this assignment.
You should present at least four independent published sources of information (textbooks, journal articles, reviews etc.) excluding websites. You can only include a web site if you have used it as a source of a diagram, and this should be correctly listed in your reference list but is not counted towards your minimum of four information sources.
To avoid inappropriate text matches with your references and to ensure a correct word count, Turnitin will be configured to ignore text in your reference list. This requires a heading called ‘References’ (without the inverted commas) immediately above your reference list. Also check your spelling of this heading or Turnitin won’t recognise it.
Criteria for assessment of your written report
The marking rubric below shows in grid-format the criteria that will be used to allocate marks for your report – content (40%), presentation (30%) and referencing (30%).
Criterion
Fail
(less than 50%)
Pass (D)
(50 – 60%)
Good (C to B)
(60 – 80%)
Excellent (A)
(80 – 100%)
Content
(40%)
Protein & ligand, their interaction, biological function (points 1-5 as given)
Plus at least 1 other feature presented
Does not meet minimum standard.
Covered bare minimum (points 1-5 only)
Covered minimum with some further explanations in one area
Covered minimum with further explanations in most areas and excellent explanations in at least one area.
Presentation
(30%)
– Clear, concise and coherent presentation style
– Complete grammatically-correct sentences and appropriate use of paragraphs and/or headings
– Logical layout (clear and coherent flow of information)
– Diagrams – appropriate use of images
– Interesting
Does not meet minimum standard.
Used full sentences, organized into paragraphs.
Spelling substantially correct.
Figures included and referenced.
Grammatically correct sentences.
Scientific vocabulary used appropriately.
Writing had a logical flow and a conclusion.
Figures included with titles and referenced.
Grammatically correct with a logical flow.
Scientific vocabulary and concepts used correctly and with understanding.
Figures with explanatory title and legend and referred to in the text.
Shows understanding of whole topic.
Referencing
(30%)
– Gathered at least four references / sources (no web sites)
– Correct use of in-text citations
– Full references provided appropriately in reference list
– Assessor to analyse Turnitin Originality Report
Does not meet minimum standard.
Four references from textbooks, for example.
No web sites (*except for diagrams only).
References linked to text with in-text citations.
Reference list complete.
Four references including journal review or article, for example.
No web sites (* except for diagrams only).
References linked to text with in-text citations.
Reference list complete with consistent formatting.
More than four references, including journal reviews or articles.
No web sites (*except for diagrams only).
References linked to text with in-text citation and figure legends.
Reference list complete with consistent and correct formatting and in-text referencing used appropriately
REFERENCING
Whenever you produce any written document which includes ideas that are not your own, or information from another source, it is important that you give due credit to the person(s) who produced the original information. You acknowledge your sources through referencing throughout your document. As well as giving credit to the source of your information, a reference list or bibliography shows how widely you have read, enables readers to check your ideas and follow up your sources and importantly shows the difference between your ideas and those of others. The important things about referencing are that:
(i) it acknowledges the original source of the information;
(ii) it enables the reader to source the original information if required; and
(iii) it is of a consistent format within a document (for clarity).
There are basically two main styles of referencing, often referred to as the Vancouver (numerical) and the Harvard (author-date) systems. Each system has its pros and cons, but in science the Harvard system is generally preferred for several reasons.
A ‘Harvard’ referencing style simply requires that the author(s) name(s) and the date of publication be given at the appropriate point in the text using an ‘in-text citation’ and that a reference list is provided at the end of the document, in alphabetical order based on first author surname (then sorted by date of publication if more than one resource is written by the same author). A Harvard referencing style demands nothing more than this.
Within the Harvard family of styles are dozens (hundreds?) of variants with minor formatting differences which frankly make no difference to the functionality of the style. The College of Science Health & Engineering (CSHE) ‘First Year Survival Guide’ provided to many of you last year stated that in the majority of first year subjects you would be expected to follow the APA (American Psychological Association) style of referencing, but that in second year and beyond you may be required to use different styles in different subjects, reflecting the real-world usage of myriad referencing styles.
Bibliography or reference list:
What’s the difference?
Many people use these terms interchangeably, however they are not the same. A bibliography is a list of works used as sources of information when preparing your text, whereas a Reference List is the list of sources from which you have taken information directly.
The referencing style to be used in this BCH2IBM assignment is the Harvard (author-date) system. The APA style is just one of many examples of the Harvard family of referencing styles, so you are welcome to use it for this assignment if you wish.
For further information on referencing systems and style formats, refer to the guides available through the library at: http://www.lib.latrobe.edu.au/referencing-tool.
In-text Citations
In your text you must include references to all sources of information used for the content of your work. To identify references, in-text citations should be given as the last name of the author and the year of publication. Page numbers to direct the reader to specific information must be used for direct quotations and for referring to specific data or figures.
Format of in-text citations
Your reference has….
In the text you write…
Example
one author
authors surname and the year
GFP was used to study GPCR regulation (Milligan, 1999).
Milligan (1999), showed that GFP …
two authors
both surnames and the year
mGluRs have a 7 trans-membrane spanning structure (Pin and Duvoisin, 1995)
three or more authors
first authors surname followed by ‘et al.’* and the year
BAY36-7620 acts as an inverse agonist (Carroll et al., 2001).
A study by Carroll et al. (2001), showed that…
multiple references
chronological order, with those in the same year alphabetically by author surname
mGluRs have a 7 trans-membrane spanning structure (Sladeczek et al., 1992; Pin et al., 1994; Pin and Duvoisin, 1995; Pin et al., 1995; Bockaert and Pin, 1999)
multiple references – same author and year
distinguish by lower case letters “a”, “b” etc.
mGluRs have a 7 trans-membrane spanning structure (Pin et al., 1995a; Pin et al.,1995b
you wish to cite a reference within a reference you have read (known as a secondary citation)
you have not seen the original source material, but have read about it in another reference.
Original authors name and year followed by “cited in” the source you have read name and year
mGlu1a receptors display agonist-independent activity (Prezeau et al., 1996 cited in Carroll et al., 2001).
Prezeau et al. (1996; cited in Carroll et al., 2001), show that mGlu1a receptors display agonist-independent activity.
* ‘et al.’ is an abbreviation of the Latin ‘et alii.’, meaning ‘and others’
Format for references in your reference list
Book
• author’s surname(s) and initials
• year of publication
• title of book
• edition (if applicable)
• place of publication
• publisher
eg. Nelson D.L. and Cox M.M. (2012) Lehninger: Principles of Biochemistry, 6th edn. W.H. Freeman and Co., New York.
Chapter / article in an edited book
• author’s surname(s) and initials
• year of publication
• title of chapter
• editor*
• title of book
• edition (if applicable)
• place of publication
• publisher
• page numbers of chapter
eg. Prezeau L., Parmentier M.L., Carroll F., Pin J-P. (2003) Inverse agonists to explore the mechanisms of metabotropic glutamate receptor activity, in AP IJzerman (ed), Inverse Agonism: Proceedings of the Esteve Foundation Symposium X, The Netherlands, Elsevier Health Sciences, pp235-244.
Journal articles
• author’s surname(s) and initials
• year of publication
• title of article
• journal title** (in italics)
• journal volume (in bold)
• page numbers
**You may use the full or the abbreviated journal title; however it is important to be consistent, i.e. use the full journal title every time or the abbreviated title every time.
The full titles and official abbreviated titles for journals listed in Medline are easily found at: http://icmr.nic.in/health/Medline2007.pdf.
* preceding the editors name, type the word ‘in’ to indicate that the chapter title is in the book. After the editors name, type ‘(ed)’ or ‘(eds)’ to indicate that they are the book editors
Eg. Carroll F.Y., Stolle A., Beart P.M., Voerste A., Brabet I., Mauler F., Joly C., Antonicek H., Bockaert J., Muller T., Pin J-P., Prezeau L. (2001) BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity. Mol Pharmacol. 59, 965-73.
Internet references
As previously noted, for this BCH2IBM research assignment you may not use internet sources as formal references. During BCH2MBC in second semester, we will run an information session on evaluating the validity and integrity of material sourced from the internet, after which we hope that you will be able to use web sources judiciously.
For your protein-ligand interaction research assignment, we have made one exception – you can present figures (diagrams, graphs etc.) that you find on the web if you wish to.
Note: Many of the pictures you find on the internet have in fact been reproduced from a published source, most often text books and journal articles. It is always better to provide the reference of the original published source than an internet site on which you may have seen a copy of the item. If you feel that the best figure for your report is only available on the internet, please provide the author and date of publication in text (if possible; you must provide sufficient information in-text for the complete reference to be found in your reference list) and the following information in the reference list:
• author’s name (if known)
• date of publication or last revision
• title of document
• title of complete work (if relevant)
• URL, in angle brackets (<>)
• date of access
The information required for citing material from the internet does not vary from print material. You still need to include, where possible, an author or organisation responsible for the material, a title, a date of publication, a place of publication, the publisher and the ‘extent’ of the item (number of pages).
Example : Information from web pages
Darby, M. [internet] Bile Duct Cancer, 31 March, 2007, Cancer Council Victoria, cited 22 February, 2011. Available from:
http://www.cancervic.org.au/about-cancer/cancer_types/bile_duct_cancer
Journal Articles and the Internet
Many publishers of printed journals now also provide their journal articles electronically on line. Students are often confused about whether to cite journal articles that they may obtain from the internet as an electronic web-based source or not. As a general rule, regardless of whether you downloaded the article from the internet or not, if the article is available in a print journal then you should reference it as a print journal as previously described. However, many journal publishers now also provide additional material which is available only through their electronic media, not in the printed journal, which complicates this issue. So generally you should cite the printed journal article if at all possible, but if you use a figure or cite material that is only available electronically, then you should indicate this by placing the source [internet], and the date cited, after the journal title. You also need to include either the URL (universal resource locator) or the DOI (digital object identifier).
Example
Austgen, K., Johnson, E.T., Park, T-J., Curran, T., & Oakes, S.A. (2012). The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress. Nature Cell Biology, [internet supplementary information] [cited January 13th 2012] 14 (1), 87-92. doi:10.1038/ncb2395
Pre-print articles
If you are reading very recent material, you may find that there are no volume or page numbers provided for a journal article. This is because many journals will publish accepted articles electronically prior to the formal publication of the print journal. These articles will generally have a digital object idenitifier (DOI) which needs to be included in the reference until the print volume details are available. The DOI does not need to be included if the volume and page numbers are available and included.
Example
Droese, S., Bleier, L. and Brandt, U. (2011) A common mechanism links differently acting complex II inhibitors to cardioprotection: modulation of mitochondrial reactive oxygen species production Mol Pharmacol mol.110.070342; published ahead of print January 28, 2011, doi:10.1124/mol.110.070342
TURNITIN
La Trobe University subscribes to a web-based text-matching facility called Turnitin which can be used by students and staff to check for correct citations and to help eradicate the inappropriate reproduction of other peoples work without proper acknowledgment (i.e. to avoid plagiarism). We expect that you will take the time to familiarise yourself with the program and we hope that you will use it routinely to monitor the originality of your work.
Turnitin scans through the text of a submitted document and compares that text with material held in its databases. The huge repository of text held by the Turnitin system includes material published in journals, books and textbooks, material available on the web together with assignments and other written work submitted by past and present students worldwide. Text in the submitted document that is found to match any stored text is detected and highlighted, and the source of the matching material is also identified. This process enables you, the student, to check whether you have paraphrased appropriately or if you should use direct quotations etc. It also allows teaching staff to check for plagiarism, including copying between students.
You must run your report on the ‘Protein-Ligand Interactions’ Integrated Assignment through the Turnitin program, in fact it is only through the Turnitin system that you can submit your work for assessment.
Using Turnitin
Click on Turnitin link in the ‘Integrated Research Assignment’ section on the BCH2IBM LMS site to be directed to the Turnitin submission pages for multiple drafts and the single final version of your assignment report. This link will open a few weeks before the first due date for this assignment.
You must submit your report to Turnitin using this Turnitin link. By doing so, the LMS automatically informs the Turnitin system of your identity (i.e. the fact that you are a La Trobe University student, your name, your Student ID number and that this assignment is for BCH2IBM). It is essential that you are logged in to the LMS under your own name when you submit work to Turnitin. Do not log onto the Turnitin system using any other method, or using anyone else’s log-in credentials.
Note: If at any time during this procedure a message appears on your screen warning you that a

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