# BISC209/S13:Guidelines for writing a scientific research report

Wellesley College-BISC 209 Microbiology -Spring 2013

## Guidelines for Scientific Writing

Most scientific work is published in a specific format called scientific style. Although there are many small differences among science publications, a standard format has evolved so that readers know where to find specific categories of information (i.e., hypotheses, background and pertinent literature, methods, implications of results). Use of the standard format is a very important part of scientific writing. Arbitrary modifications of the format by "creative" writers can only confuse readers and lessen the paper’s clarity and impact. The best way to learn this type of writing is to read good examples of scientific writing in journals and to practice. Here are some guidelines for writing your papers for this course in scientific style.

All sections of a paper should be labeled, except for the title and introduction. Double space all sections, except for the abstract and references.

## A.) Title

Compose a title that reflects the major conclusion of the paper. Remember that “the story is about who shows up first”, therefore, you should stress the answer to the experimental question over the experimental tools. Generally, the title should be less than ten words.
Compare these three examples:
"Response of patients to different doses of ice cream"
"Frequent administration of ice cream boosts patients' morale"
“Morale in hospital patients is improved by serving more ice cream”
The first example gives little information about the findings. The second is unclear: we don’t know how the ice cream was used (“applied” doesn’t necessarily mean “eaten”) or enough about the patients. It appears, in the second example, that the topic is uses of ice cream rather than about how to boost patient morale. Although the third example is more than ten words (11) and it is in passive rather than active voice, its advantages are that it’s clear that the main topic is hospital patient moral and that the variable showing positive correlation is eating ice cream. The third title, therefore, is preferred.

## B.) Abstract

The abstract is a brief summary (~200 words in a single paragraph) of the entire paper. It should include, in this order, 1) the topic or hypothesis driven question and a brief explanation of its importance, 2) a brief, general summary of methods used to address the question experimentally, 3) the specific, major findings of the study, 4) the conclusions’ significance. The abstract should “stand alone”, meaning that it is an independent section, used by readers to decide whether or not to read the paper. All information in the abstract must also be found in the body of the paper. The abstract is written last because it is far easier to summarize a completed paper than an intended one.

## C.) Introduction

This section introduces the reader to the subject, the topic, and the experimental design that will test the hypothesis. It includes background information that explains the rationale for the hypothesis and gives the context of the experimental question. It is important to make your topic clear early in your paper. A captivating first sentence might identify a controversy or pose a question of general interest. It should entice your audience to continue reading. Students often begin with, “The purpose of this lab was…” or “In this experiment we…”. Avoid both of these trite or inappropriate beginnings. Vary your sentence structure to avoid the expected. Make the first sentence show that there is a broad context of interest for the narrower question that you will explore experimentally. Remember that you are writing in the style of a scientific original research report, not a lab assignment; therefore, you should describe the entirety of your work as a “study” rather than as an “experiment”. Your investigation will, in all probability, include several experiments.

Following your carefully considered, compelling topic sentence, give a brief history of the topic (moving from older to newer information) to justify your study and to show how your study will add to the body of scientific knowledge on the topic. Be selective and brief. Avoid misleading your reader as to the study’s direction by including only the most relevant previous findings. Use in-text citation of the background studies or findings that you mention.

The introduction includes other general information that your audience needs in order to understand the experimental question addressed, how you formed your hypothesis, and how you will attempt to answer the question experimentally. Because the background information is the rationale for your hypothesis, the hypothesis should follow logically from that flow of information, but it does not have to be directly stated. Avoid the statement, “the hypothesis is….”. Instead, you might say, “ because…. and…, it is expected that….”.

The introduction’s concluding paragraph should be limited to a clear, concise restatement of the experimental question(s) and the general approach that will be used to test the hypothesis. Detailed information about methods is more appropriate for the Methods section. Save your results and conclusions for later in the paper.

Basics of In-Text Citations:
Because your introduction’s history of the topic should include relevant findings from previous studies, you must cite those sources using in-text citations. Proper credit must be given unless a finding or concept is so well established that it is considered “general knowledge”. How do you know which information is “general knowledge”? The rule of thumb is if basic information is available in a textbook without source citation, it does not need to be cited in your paper either; however, when you refer to specific, published research findings, those require proper in-text citation.

Direct copying of text from other sources, even if cited, is considered plagiarism. For more information about what constitutes plagiarism, please check the Wellesley College Honor Code Council web site.

Although there are almost as many different ways to cite sources are there are science journals, in this course we will use the format of the science journal Cell. It’s a version of the Name/Year citation style.

The Wellesley College library has an electronic subscription to Cell through the e-database ScienceDirect:(http://luna.wellesley.edu/search~S3?/tCell/tcell/1,129,147,B/l856~b2338006&FF=tcell&1,1,,1,0); therefore, you can use articles in a recent issue as models for how to format properly your paper’s references. Alternatively, you can instruct a reference management software program such as EndNote™ to automatically format your references in this style.

Name/Year Cell style in-text citation style:
Cite a single author source at the end of the sentence as (Beers, 2007); if there are two authors (Beers and Kemphues, 2007) or three or more authors (Beers et al., 2007). If you refer to the authors in the text by name, for example, "In a study by Beers and colleagues (2007) ...”or “Beers and Kemphues (2007) found …”, it is only the year that is cited, directly following the authors names . You need not give the (Name/Year) citation again the end of the sentence. Note that unlike other common citation formats, Name/Year style does not include page numbers. That information is included with the full citation found in the Reference page at the end of the paper. See section H here and/or refer to recent published papers in Cell to see how to format the Reference page.

## D.) Methods

Students commonly have trouble with the methods section, primarily because it is mistakenly considered to be the place to describe exactly what you did in lab, step by step. Instead, think of this section as the place to present the progress of the experiment through a detailed but brief description of the methods used to collect and to analyze your data. Present enough information so that the reader can evaluate what you did, see how you achieved your experimental goals, and could, possibly, replicate your work. Do not clutter your paper with trivial or nonessential information. This section should not be a transcription of the lab manual or a chronological description of your lab experience. It should be as succinct as possible.

Divide the Methods section into subheadings with titles that give the goal as well as the main tool used to achieve the goal of each part of the experiment, e.g, “DNA fragmentation and separation by restriction enzyme digestion and agarose gel electrophoresis” is a better subheading than “Gel Electrophoresis” or “Identifying the Gene”.

Methods are always presented in the past tense because the experiments are complete. Third person is preferred, even if it requires passive voice, because avoiding first person helps you focus on the progress of the experiment rather than on your lab day (“DNA was extracted in 70% ethanol” is preferred over, “We added 1ml of 95% ethanol to a tube containing…”).

Never use the word “tube” (or similar words such as “plates”). Instead of stressing the container, explain what’s in it, using specific terms that increase understanding. You reader doesn’t want to know all about your lab day, but she is struggling to understand how you reached your goal from the starting materials. Rather than giving a recipe (e.g. mixed this vol. of this with this vol. of that), give effective concentrations of reagents when you describe crucial points in the experiment.
Example: “Product formation after 10 minutes was halted with the addition of 0.1M Na2CO3 to each enzyme/substrate reaction”, is preferred over “1ml of 1M Na2CO3 was added to each tube in 30 sec. intervals.”

How to calculate effective concentration:
You should include ingredients and concentrations of all reagents at first mention, if you can. The ingredients and concentration of all stock solutions, suspensions, and reagents (or where to buy proprietary reagents) are found in the lab protocols or in a separate appendix. Give reagent “recipes” or supplier information in parenthesis after you first mention anything that will have to be made or bought. Many reagents are proprietary; therefore, showing your reader where to buy them is the best you can do if you can’t give enough information for your reader to be able to make them.
Examples: Cells were cultured at 37C overnight in Luria broth (1% tyrptone, 0.5% yeast extract,1% NaCl ).
DNA was extracted from leaf disks using a Hot Start® DNA extraction kit available from Invitrogen, Inc.

To avoid ambiguity, most scientific papers provide the effective concentration of reagents at the time of the reaction rather than giving volumes of various stock solutions as they are added together. Remember that you do not usually have to provide the effective concentrations of diluents.

Examples:

1. The effective concentration of ampicillin in broth during cell growth is calculated by multiplying the stock concentration (100 mg/ml) by the dilution created when 10 µl of the stock ampicillin was added to 10 ml (10,000 µl) of broth. In this case, the effective concentration of ampicillin in the culture is 0.1 mg/ml AMP because there is 10 µl of stock ampicillin in 10,000 µl (10 ml). This constitutes a 1/1000 dilution of the 100 mg/ml stock. Note that the concentrations of the components of the broth are not significantly changed by the addition of this small amount (10 µl) of ampicillin to 10,000 µl of LB, so the concentrations of the ingredients used to make the broth can be given as stock concentrations without adjustment.
2. If 100 µl of a solution containing 50 mM glucose, 10 mM EDTA and 25 mM Tris buffer was used to resuspend a bacterial cell pellet, the concentrations in the stock solution are not altered because the pelleted cells do not represent a measurable fluid volume.
3. When 200 µl of a stock solution, containing 0.2 M NaOH and 1% SDS, are added to 100 µl of suspended cells, the total volume is now 300 µl. The diluted solution contains effective concentrations of 0.132 M NaOH and 0.66% SDS. These effective concentrations were calculated by multiplying the stock concentrations by the fraction of the volume of the NaOH/SDS solution to the total reaction volume: 200 µl/300 µl = 2/3 or 0.66.

Sample partial Methods section:
Restriction Enzyme Digestion and separation of plasmid fragments by electrophoresis
Plasmid DNA was digested with a restriction enzyme by combining 5ng plasmid pHM64 (Table1) and 5ng plasmid p280Δ-35 (Table 1) with 1µl HindIII ( SigmalAdrich www.sigmaaldrich.com) for 20 min at 37C in High Salt Buffer (0.1M NaCl, 50 mM Tris pH 7.5, 10mM MgCl2, and 1mM DTT). The DNA fragments from the digestion were separated by agarose gel electrophoresis by applying all of the digested DNA to a 1.5% agarose gel in TGE (0.25M Tris, 1.9M Glycine, 13mM EDTA) with 0.5% SYBR safe DNA stain from Invitrogen www.invitrogen.com. DNA fragments were subjected to electrophoresis at 100V for one hour. Stained and separated DNA fragments were visualized and photographed under UV light.

If you use mathematical formulas or statistical analyses, include an explanation of those calculations or statistical tools. Explain the variables that were compared, the types of statistical tests used, and for what they were used. If you calculate a p value, include the α-level (threshold significance probability—often 0.05). Explain how measurements (original data collected) are transformed, perhaps by taking their logarithms, multiplying by a dilution factor, using a molar extinction co-efficient to transform Absorbance to concentration, etc.

Example of how you might write about a statistical analysis tool or a mathematical transformation from Absorbance to enzyme activity in your Methods section:
Statistical Analysis:
Averages and standard deviations of both the five and ten week counts were calculated and graphed as a column graph. A one-way ANOVA and Tukey HSD test were performed on both counts to compare the yeast/whole wheat combinations. The growth rates between 0-5 and 5-10 weeks were calculated and graphed as a linear plot in Excel. Student’s t-tests were run to compare the 0-5 and 5-10 week growth rates for all yeast/whole-wheat combinations.

Mathematical Transformation:
Intensity of yellow color was determined as A420nm in a Hitachi spectrophotometer and converted to beta-galactosidase activity units using the following formula: $\displaystyle{ Activity Units = 1000x (A420-(1.75 x A550)/time x volume x A600) }$
where time (10)is in minutes, volume (0.75)is in mL of reacted lysates, 1.75 is the Molar extinction co-efficient of ONP, A600 represents the absorbance reading at 600nm of 1:10 diluted unreacted, whole cells (to account for differences in cell density) and A550 is measured in the halted reactions at the same time as A420nm measurements were taken( to account for turbidity from cell debris).

## F.) Results

The results section should begin by framing the overall investigation and its goals. The results section then leads your reader through your experiments in a narrative that describes how the data lead to the conclusions of the paper. This section should NOT consist only of figures and tables. Each paragraph should begin with a topic sentence that frames a particular experiment, addressing the biology you are exploring in it. The narrative portion is always written in the past tense because the experiments you describe are complete. There should be proper transitions between paragraphs and varied sentence structure as you describe the experiments (without excessive methods details). There must be a specific reference to every figure or table included as you emphasize the main findings of each experiment. A good template to use for the overall structure of this section is, “Why, How, Where, What, So What?”.

Example: “To determine whether or not cheek epithelial cells contained a nucleus (WHY?), they were stained with methyl green, which binds to acidic molecules such as DNA (WHAT?). As shown in Figure 1 (WHERE?), one round, dense, darker-staining structure of diameter 5-10mm was seen in each cell (WHAT?), suggesting that these cells do indeed have nuclei (SO WHAT?).

The correct way to refer to a table/figure in the narrative part of Results is:
Length of activity periods was inversely proportional to temperature (Table 1).
Parents of both species fed their chicks between 09:00 and 14:00 (Fig. 1).

Using Statistical Analyses in Results:
Your results must include a few sentences that specifically describe any statistical tests’ results and their interpretation. It is important to focus, primarily, on the overall trends of your data, when reporting the results of statistical analyses. This information—the direction and magnitude of differences among treatment groups--is the key outcome. Remember that a test for significance merely indicates the probability of a given outcome happening by chance.
Useful tip: Use the term “significant” only when referring to the results of a statistical test for significance. This word has a very specific meaning in a scientific paper.

Examples are given below for ways to interpret the statistical tool and include the evidence that allows the conclusion. Note in the examples that the statistical tests are presented in the following order: name of the test, test statistic, degrees of freedom (or sample sizes of the groups tested), P value, and alpha level. This form may be slightly modified for different types of tests but all four parts of this information should be included.

Examples:
For a Chi-square test:
"Significantly more honeybees visited inflorescences with petals than without petals.
(Chi-square test, X2 = 7.20, df = 1, P < 0.05, α = 0.05)."

For a Student t-test:
“The mean number of beetles in population E was significantly higher than in population D
(t-test, tstat = 2.87, df = 8, p = 0.02, α = 0.05).”

For an ANOVA or Tukey statistical test:
“Mean population sizes in corn or white flour were not significantly different from each other but were significantly different from the mean population size in wheat flour (ANOVA, F = 22.7, df = 2, 6, p = 0.002, α = 0.05; Tukey HSD test).”

Wilcoxon test:
Show it similarly to the Chi square as: Wilcoxon test, X2 = 1.82, df = 1, P = 0.61, α = 0.05

Tables and Figures:
The narrative text of Results describes your findings while the data that documents those findings is processed and presented in tables and figures. An effective table or figure should stand alone, meaning that the reader should be able to understand, generally, the experiment and the data’s meaning without reading either the Methods section or the narrative description. Unprocessed, raw data are usually not included in the body of the paper. Instead, process and present the data in a way that makes your main findings readily interpretable by someone who was not in lab. Number figures and tables separately and consecutively in the order to which you refer to them in the text.

Because it should be possible to understand the main point made by each figure or table’s data without reading the results narrative, table headings and figure captions must include all of the information needed to interpret the data presented; however you should not include in figure legends or table headings a discussion of your findings. In addition, make sure you reduce substantially (compared to the level of detail found in your methods section) the description of the data collection.

Figures:
Any graph, map, photograph, or drawing is a figure. Label the axes carefully with titles and specify the units.

Figure 1 Protein calibration curve. A range of concentrations of bovine serum albumin (BSA) were reacted with biuret reagent (alkaline copper sulfate). The resulting change to purple color was measured as Absorbance in a spectrophotometer at a wavelength of 540 nm. A540nm values were plotted against known BSA concentrations as a regression line using Microsoft Excel in order to create a standard curve for converting A540nm measurements of unknowns to protein concentrations.

Tables:
A table is any data presented in tabular form. Each column of data should have a heading that describes completely what is in that column, including the units of measurement. Some columns may need a double heading. For example, when reporting data from different species, you should specify which column of data was collected from each species. It will also be necessary to give an additional heading over all of the species’ data columns indicating what type of data are in these columns. Enter data values so that decimal points are vertically aligned, and align and space all entries for maximum clarity. Note that Table headings are positioned above the tabulated data while figure captions are found below the figure. Footnotes are used when symbols or abbreviations require explanation.

Table example:
Table 1. Means ± SE for a test of the hypothesis that food type affects flour beetle (Tribolium castaneum) population growth in a 70-d period. ANOVA, F = 22.7, df = 2, 6, p=0.002, &#945= 0.05. Tukey levels1 not sharing the same letter are significantly different.

Type of flour Number of Beetles
Mean ± SE
Tukey Level
Corn 196 ± 13.3 B
Wheat 385 ± 17.3 A
White 132 ± 42.1 B

1 Tukey HSD test

## G.) Discussion

If you include suggestions for improving your study, always present those ideas in a positive way; do not excessively denigrate your results or your experimental design. For example, a statement like "sample sizes were too small to draw conclusions" causes the reader to ask why larger sample sizes were not gathered, and it makes her question your ability to think creatively; were no conclusions possible?

Emphasize the “take-home’ messages of the paper in a concluding paragraph. Your conclusion should summarize the principal findings, interpretations, and implications of your work.

## H.) References or Literature Cited

This section is a list of all articles cited in your paper in a specific format that must be followed exactly. There are almost as many formats as there are science journals; therefore, you will have to pay careful attention to directions of your instructor and use a style guide, models or a reference management software program such as EndNote™ to get this section exactly right. Your references page may be called Literature Cited or References, depending on the specifications of the citation style you are asked to use. In BISC110 you are using the citation style of the journal Cell; therefore, your cited source list is titled “References”, rather than Literature Cited or Bibliography. A bibliography is a list of possible references on a subject, including some that are not used specifically in the paper. Your reference list will include only those papers specifically cited in your paper. Footnotes are not used in citations in scientific format; the cited sources are found at the end of the body of the paper.

Lab Manual
The lab manual should not be cited as a reference. It is only available to students in the course and therefore a reader could not verify the information.

Websites
Websites may be used as starting points for your investigation but MOST should not be used as sources to support your conclusions. Sites like Wikipedia are good starting points for investigating a general topic; however, the information is not verified. Please find a verified source to support any information from these sites. A few exceptions to this rule include government sponsored data bases such as the Center for Disease Control (CDC) or the National Institute of Health (NIH) web sites.

Personal Communications
Avoid or limit “personal communication” citations in your text. These are generally used when you are using someone else’s unpublished experimental data to support your conclusions. Information presented by your instructor should not be cited as a personal communication– find and cite a published, primary literature source.

Your cited sources list is arranged alphabetically by the first author's last name. The format examples shown below are from the journal Cell, the format to be used for this course. Please note that each journal, course and/or instructor may require a specific format that may be slightly different than the one provided below. Be sure to follow the guidelines for references set by your course instructor.

References to journal articles:
Black, D.L. (2003). Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336.

Carissimi, C., Saieva, L., Baccon, J., Chiarella, P., Maiolica, A. , Sawyer, A., Rappsilber, J. and Pellizzoni, L. (2006). Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly. J. Biol. Chem. 281, 8126–8134.

Will, C.L. and Luhrmann, R. (2001). Spliceosomal UsnRNP biogenesis, structure and function, Curr. Opin. Cell Biol. 13, 290–301.

Reference to a paper in an edited volume (e.g., where separate authors write chapters of a book): Riddiford, L.M. (1993). Hormones and Drosophila Development. In: Bate, M. and Martinez-Arias, A., Editors, The development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 899–940.

Reference to a book:
King, R. C.(1970) Ovarian Development in Drosophila melanogaster. Academic Press, San Diego, CA. 789-790.

## Erata

1. Dates - day comes first, then month, and year: 18 July 1977
2. Time - use the military system: 08:00 is 8 A.M. and 15:00 is 3 P.M.
3. The first time you mention the common name of a species or taxa in the text of your paper you must give the complete scientific name also, i.e., white oak (Quercus alba) or bumblebees (Bombus spp.). Remember that genus names start with an uppercase letter, but species are written in lower case. Both should be underlined or italicized.
4. After the first use, you may use either the scientific or common name or an acronym, e.g. Escherischia coli first, then E. coli. This rule also applies to other commonly used acronyms, such as, polymerase chain reaction (PCR) or long chemical names. Introduce both the full term and its acronym on first mention; subsequently, the acronym is sufficient. Because the abstract is an independent, brief report of the entire scientific paper, terms introduced in the abstract must be reintroduced in the body of the paper. Thus full scientific names and acronyms will be given at least twice in your paper--once in the abstract and once elsewhere within the paper. Both can be given parenthetically, such as: gray catbird (Dumetella carolinensis), raspberry (Rubus spp.), or 2,6 dichlorophenol indophenol (DCPIP) # Numbers over ten are presented as numerals; spell the numbers ten and under. Exceptions to this rule are:
1. When presenting measurements, you can always use numerals, e.g., 6 mm.
2. Dates, percentages, page numbers, figure and title numbers, and decimals can be given as numerals.
3. When giving a list, with some numbers included that are over ten, all can be given as numerals: 18 cardinals, 12 blue jays, and 5 crows.
5. Always use the metric system.

## Writing Tips

1. Be sure to divide the text into appropriate paragraphs. Each paragraph must begin with a topic sentence that gives the paragraph’s context (connects backwards to the previous paragraph) and a concluding sentence that describes the main point of the paragraph. Remember that the end of a paragraph or a sentence is the stress position. Don’t confuse your reader by putting the most important information in a non-stress position or unimportant information in the stress position. Information should move, generally, from old to new.
2. You should, largely, write about your study in past tense; however, it is common to write about generally accepted scientific theory worked out by others in present tense. Active voice is generally more interesting than passive; although, in science writing, passive voice is sometimes preferred (particularly in the methods section) because it seems more objective and it appropriately changes the focus from the experimenter to the experiment.
3. Remember your goal is to convey information; you must write so that you cannot be misunderstood. Readers have structural expectations of a paper in scientific format and it confuses them when those expectations aren’t met.
4. Eliminate redundancy. Decide where a detailed explanation of a concept would be most helpful to your reader; limit yourself to a brief summary when mentioning that concept elsewhere in the paper.
5. Prepositions should have objects: Wrong: Samples A-F are shown in the plots they were taken from; Right: Samples A-F are shown in the plots from which they were taken.
6. The word 'data' is plural; 'datum' is singular. Use the verb with proper agreement: “data are shown” as…
7. Be sure that pronouns refer clearly to their appropriate antecedent. Example: "Sometimes nests were built in bushes, and they were often hard to find." Does 'they' refer to the nests or to the bushes?
8. Avoid anthropomorphism (giving human characteristics to non-human subjects.) Examples:
1. A plant chooses to grow in a wet area.
2. Honey bees have a visual appreciation for color
3. Honey bees try to optimize costs.
4. Wind discouraged pollinators.
9. 'Species' is both singular and plural (a 'specie' is a coin).
10. Do not use slang, colloquial language, or idiomatic phrases. “Ran a gel” is colloquial.
11. Contractions should be avoided.
12. Avoid use of qualitative and often meaningless adverbs such as almost, largely, very, most, strongly, greater, etc. Replace such terms with quantitative evidence or an example that supplies context. Example: Which is a more persuasive statement?
1. Facebook is a wildly popular online social networking tool...
2. Facebook is the Internet site most frequently viewed by males and females, aged 17-25, in the United States today (eMarketer, 2007). Sixty-nine percent of females and 56% of males in that age group have Facebook accounts; nearly 65% of users log on to Facebook once a day.
13. General statements like “results indicate” or “data show” are too ambiguous. Exactly which measurement or comparison or statistic leads to a conclusion?
14. Writing concisely means eliminating unnecessary words. Often, even a 'the' is unnecessary. Wordy: The areas without trees had a greater density of the defoliated plants. More Concise: Areas without trees had a greater density of defoliated plants.

Clarity in writing is a fundamental goal. Scientific writing should not be flowery or use vocabulary that is unintelligible to its target audience. Explain terms and concepts that your audience isn’t likely to know or use simpler language, as long as it’s accurate. Use the correct word. Although proteins and genes are related, they are not the same thing. Write not only to be understood, but so that you cannot possibly be misunderstood.

There are a myriad of small and large concepts to remember when structuring a paper in scientific format, especially if this style of writing is new to you. Each time you write or revise a paper you should review these guidelines. Before submitting a paper, reread it critically several times for clarity, conciseness, and grammatical errors.

An excellent discussion of structure and clarity in science writing is found in an article, “The Science of Scientific Writing” by George D. Gopen and Judith A. Swan in the American Scientist (Nov.-Dec. 1999), Vol. 78, 550-558.

Often you can answer your own questions by looking at models in published journal articles.