Dr. Daniel McGowan 论文写作系列第五讲Good study design and forward planning如何做好研究设计和预先规划工作Rejection following peer review can mean a considerable amount of additional work for many authors to get their studies published. In the worst cases, their studies may be simplyun-publishable. Much heartbreak and hard work can be avoided by simply planning and designing your study properly in advance. In the long run, this will save you time, allowing you to get on with the research for your next big paper.No-one wants to have to repeat experiments because the controls were inappropriate or the case/sample numbers were insufficient to provide enough statistical power. Frequently though, researchers rush into experiments without making all the proper considerations, and this can result in delays when their manuscripts reach the peer review stage. Remembering a few basic principles of study design can help to reduce the risk of outright rejection and repeated experimentation.1. Have a hypothesis or research questionHaving a hypothesis or appropriate research question enables you to …frame‟ your research within an appropriate context, which in turn will help you apply the appropriate controls. It will also help you describe the rationale for your study when it is time to write it up. Having a hypothesis also means that the objectives of the study are clearly defined, thus reducing the chance that your study will be open-ended and possibly criticised for being incomplete. You can then logically work through these objectives and, importantly, present your results in a logical manner rather than haphazardly.2. Ensure that the appropriate methods are usedOnce you have a clear idea of the aims of your study, and the specific research question you are setting out to answer, you will need able to determine what methods would be appropriate to achieve these. Important considerations include deciding whether subjective, qualitative data will be sufficient to address your question, or whether there is a need for more quantitative methods. For basic science studies, such considerations might include the following questions. Will the combination of RT-PCR and in situ data be enough, or is there a need for qPCR? Is Western blotting alone sufficiently sensitive or do you need to also perform immunohistochemistry and cell counting experiments to show a difference between groups? For clinical studies, important considerations include the choice of controls, sample sizes, statistical tests and approach, all of which are described in more detail in the points below.3. Ensure that the appropriate controls are usedControls are included in experiments to rule out alternative hypotheses. There‟s an old saying that “nothing can be proven, only disproved”, and this is precisely why appropriate control s are necessary: to disprove any feasible alternative interpretations of the data you obtain and/or to eliminate or minimize the effects of extraneous variables. Consider what alternative hypotheses exist, and systematically rule them out by performing experiments that disprove them. There are generally two types of controls: positive and negative. Positive controls show that a negative result is not due to a failure of the experimental system. Negative controls provide an indication of the …background noise‟ or baseline value with which to compare values from your experimental sample. In quantitative studies, a “relative control” or “housekeeping control” is required to show that changes in the apparent levels of a target gene or protein are not caused by differences in the amounts of protein or DNA in the sample. These levels can be used as a baseline to measure changes in relative levels of a target gene or protein. Commonhousekeeping molecules include β-actin and GAPDH. In clinical trials, subjects in a placebo group in intervention trials, and normal control subjects in observational trials, need to be matched as closely as possible to those in the treatment or disease group in terms of age, sex and numerous other potential confounding factors. In randomized controlled trials, accepted procedures for assignment to groups also need to be followed (see, for example, the ICH good clinical practice guidelines at: /LOB/media/MEDIA482.pdf).4. Use sample sizes large enough to provide a definitive resultMany studies fail to achieve the desired impact or to fully support a given hypothesis because the effect is too small or the variability too large to show statistical significance. Often this can be simply overcome by increasing the sample size. However, once a study has been performed and the data analyzed, it can be impossible to go back and increase the numbers without starting all over again. For this reason, pilot studies are often performed in advance of larger scale studies. Talk to a statistician. Determine the size of the effect of your treatment and/or the variability in your population before starting large-scale studies, and use this information to determine the sample size required to give you statistical power. Doing this can save you time, money and potential disappointment later.5. Use appropriate statistical tests to analyze your dataStatistical analysis of your data is essential to show that an effect is genuine and significant. Tests of significance demonstrate the robustness of your findings, essentially showing how unlikely it is that your findings were obtained …by chance‟. Are your data continuous or discrete? Are they normally distributed or non-normally distributed? The nature of your data will determine how they should be analyzed and what tests are appropriate. If in doubt, consult a statistician who will be able to advise you on the most appropriate tests to use and what these tests indicate. Determining the right tests to use in advance will save you having to repeat youranalyses if you got it wrong first time round, with the distinct possibility that no significant effect will be observed when the appropriate tests are used. For clinical trials, the following guidelines may be useful: /LOB/media/MEDIA485.pdf.6. Remove investigator and patient biasMany experiments involve subjective measurements or assessments performed by the investigators, as opposed to objective results provided by the experimental system. If the investigator has prior knowledge of the groups to which individuals/samples belong, then investigator bias is a distinct possibility, and this can invalidate any of the findings obtained. In such cases, where the investigator is a factor inherent in the experimental system, it is essential that the investigator is …blinded‟ to the groups to which individuals or samples belong. Doing so ensures the objectivity of the findings and improves their reliability. Such blinding can refer to treatment in an intervention trial, or to assessment or interpretation of clinical findings in an observational trial. Similarly, the outcome of a treatment could be influenced if a patient knows if they are receiving a placebo or drug; such patient bias should be avoided, by blinding the patient to the nature of the treatment. Being aware of the potential for bias before commencing experimentation can again save the need for time- and resource-consuming repeats.7. Comply with ethical requirementsThere are strict regulations regarding the use of human and animal subjects, and in many countries, regarding the use of stem cells, cell lines and genetically modified materials. Failure to comply with these regulations will prevent publication of your findings and could lead to legal issues; at best, it will limit the range of journals to which you can submit your findings. Make yourself aware of these regulations before you commence your study and ensure that all requirements are complied with so you don‟t encounter problems later on. As well as ethical requirements regarding experimentation, there are also strict guidelines provided by mostjournals regarding the requirements for authorship, and these also need to be complied with. Clinical trials should comply with the Declaration of Helsinki(/e/policy/b3.htm) in addition to any local requirements. Informed consent is essential for most trials involving human subjects. Animal studies should comply with local and national regulations, although many journals are now aligning themselves with standards such as the NIH “Guidelines for the Care and Use of Animals”(/regs/guide/guide.pdf). Finally, many journals require a statement describing who gave ethical approval for the study.8. Clinical study registrationMany top-tier journals now request that prospective clinical trials involving human participants should be registered online in an accessible database. Many journals will instantly reject studies of this type that have not been registered. More information on this can be found at/faq.pdf. International clinical trial registries include the Chinese Clinical Trials Register (/), the Japanese Primary Registries Network(http://rctportal.niph.go.jp/), The International Standard Randomised Control Trial Number database (/) and Clinical (/). Registration should be done before the first participant is enrolled, but many of the databases do allow retrospective registration. However, by registering the trial once you receive ethical consent you will save time and overcome a major obstacle to publication.All studies are different and therefore have different requirements regarding appropriate study design. The points above are just a few of the important considerations that should be made prior to the commencement of experimentation, and the general principles apply to a variety of different study types. It is true that sometimes even peer review fails to detect flaws in study design, as shown, for example, in the following report on randomized controlled clinical trialspublished in Chinese journals: /content/10/1/46. However, if you want your study to stand the test of time, be published in a top-tier journal and to be widely accepted by the international research community, then planning ahead and designing your study to make it robust and reliable will only serve to save you time, money and heartbreak later on.Overcoming the language barrierDr. Daniel McGowan 论文写作系列第四讲Overcoming the language barrier: writing in English for non-native authorsDr. Daniel McGowan将向大家展示“克服语言障碍，非英语母语科研人员如何写作?”“Journal editors, overloaded with quality manuscripts, may make decisions on manuscripts based on formal criteria, like gra mmar or spelling. Don‟t get rejected for avoidable mistakes; make sure your manuscript looks perfect” (quote from a senior executive at a large international publishing house).Scientific writing is difficult enough for many authors who have English as their first language; for non-native English-speaking authors, writing a paper in English represents a massive challenge that can make or break their paper‟s chances of publication. With increased pressure on publication space and increased demands on editors‟ time many journals are introducing language screening protocols to check submissions before they reach the editor‟s desk; some editors simply choose to overlook papers that are too poorly written to consider or send for review in the knowledge that, among the submissions they receive, will be well written studies containing interesting and robust science. However, all is not lost for non-nativeEnglish-speaking authors: by being aware of some of the most common scientific writinglanguage errors and how to avoid them, you can improve the quality of your paper and increase its chances of being accepted.It is helpful to think of the writing process in the same way that you think about performing experiments; that is, the language needs to be easily and accurately understood by the reader, without multiple possible interpretations arising. In experiments, we use controls to rule out alternative hypotheses. In language, we must avoid ambiguities and unnecessary text (such as repetition and redundancies) to get our message across clearly. Scientific writing should possess what I call the three “C”s: clarity, conciseness and correctness (accuracy). The key to achieving this is to be as brief and specific as possible without omitting any details that might be essential for the reader to fully understand your meaning. In other words, say no more than you need to accurately convey your message. Although writing that fails to meet this standard is sometimes described as “sloppy” or “lazy” writing, authors are frequent ly unaware that what they have written is unclear and ambiguous. Thus, attention to detail and an appreciation of how your writing could be misinterpreted are essential. What follows is just a small selection of error types that, when present in large numbers, could result in your paper going straight to the …rejected‟ pile.Articles/Plural vs singularArticles (a/the) are adjectives that modify nouns. Where they are used incorrectly the reader can be left wondering if you are referring to a specific thing or to a non-specific item or category. Worse, they could interpret the text incorrectly and make a wrong assumption. Incorrect use of articles can also lead to confusion relating to singular vs plural senses. The word “the” should be used in conjunction with a noun referring to a particular item or group of items (it can be used with both plural and singular nouns); for example, “the sections were/the section was then stained with H&E” implies that the sections you had referred to in recent sentences weres tained. By contrast, “a” should be used in conjunction with non-specific nouns; for example, “a section was then stained” infers that a single section, any section, was stained. “A” should only be used to refer to a single item or category, and should not be used in conjunction with plural nouns; that is, “a sections” would be incorrect. Asian authors frequently leave articles out of sentences making them sound awkward and unnatural, which would be the case when omitting the “the” in “adenovirus was injected into the fourth ventricle”.1. “The antibody was injected into the hippocampus…” (articles required to specify a particular antibody, presumably already referred to in the text, and a specific hippocampus, belonging to a subject already described).2. “A new method of extraction was devised…” (“a” used rather than “the” because this statement introduces this method to the reader; therefore it is non-specific at that time. Once introduced to the reader, “the new method of extraction” should be used to refe r to that method in the specific sense).Nouns are used in the plural sense by adding an “s” to the end (in most cases). In the absence of an article, it can sometimes be unclear if the wrong sense (plural vs singular) has been used. For example, in the se ntence “Acetyl group was added”, the reader is not clear whether the author means “An acetyl group was added” or perhaps “Acetyl groups were added”. Thus, when referring to multiple items, the plural sense should be used to avoid potential confusion. This is commonly forgotten when describing figures (use “arrowheads” rather than “arrowhead” where there is more than one in the figure; likewise, use “solid bars” rather than “solid bar” when referring to a bar chart with multiple bars).1. “A biopsy was obtained…” (describing a single biopsy).2. “Biopsies were obtained from eight patients…” (no article necessary unless these biopsieshad already been introduced to the reader, in which case they would need to be referred to in the specific sense “The biopsies were obtained…”).Commas, hyphens and “which”Used incorrectly these three elements of writing can introduce ambiguities, and the potential for subsequent misunderstanding, into your writing. For example, in the sentence “Because Aβ42 levels were elevated in 75% of AD patients in studies using our method [6,7], it is critical to obtain fresh samples”, moving the comma after method to follow the word “patients” (or addition of a new comma there) would completely change the meaning. Similarly, in the phrase “calcium-induced calcium release”, omission of the hyphen completely changes the meaning of the sentence. When the hyphen is present “calcium-induced” is a compound adjective modifying the noun “calcium release”; when the hyphen is absent, “induced” is a ve rb describing the effect of calcium on calcium release. Thus, it is critically important to use hyphens with such compound adjectives to avoid misunderstandings. However, no hyphen is required to combine an adverb and an adjective; for example “highly intense staining” and “high-intensity staining” are both correct, but “highly-intense staining” is not.1. “Glutamate receptors mediated synaptic plasticity…” (tells the reader that Glu receptors are involved in the development of synaptic plasticity).2. “Glu tamate receptor-mediated synaptic plasticity…” (identifies synaptic plasticity involving Glu receptors as the subject of the sentence; note the change from plural to singular because “receptor” is being used in a general sense and not to refer to a single receptor).The word “which”, when used incorrectly, can also induce considerable confusion. It is often used incorrectly instead of “that”. Both introduce clauses that modify nouns, but “that” should be used to introduce defining or restrictive clauses and “which” should be used to introduce non-defining or non-restrictive clauses. For example, in “the sections that were positive forGFP were subjected to cell counting procedures”, the “that” introduces a defining clause that defines exactly which sections were subjected to cell counting. By contrast, in “the sections, which were positive for GFP, were subjected to cell counting procedures”, the sections that were subjected to cell counting are rather loosely defined, possibly referring to sections that have been described in the previous or recent sentences. The clause about GFP positivity provides the reader with additional information, but is not essential to understand the meaning of the sentence; that is, it is disposable. Because “which” is used in this way, writers need to ensure that it is absolutely clear what the “which” is actually referring to, possibly whatever immediately precedes it (most commonly) or possibly the main subject of the sentence. For example, the sentence “microglia migrated to the site of the lesion, which was associated with increased levels of ED-1” is somewhat vague, because it is unclear if the “which” is referring to the lesion or to the migration of microglia. If there is ever any doubt about such a sentence, it is best to re phrase it completely; for example “migration of microglia to the site of the lesion was associated with increased levels of ED-1” or “microglia migrated to the site of the lesion, and immunohistochemical analysis revealed increased levels of ED-1 at this s ite”, both of which are unambiguous.1. “Data were normalised to the housekeeping gene actin, which was used as an internal reference…” (here, the “which” refers to actin, which is therefore the subject of the following clause).2. “Data were normalised to the internal reference housekeeping gene actin, revealing increases in the levels of…” (to refer to the analyzed data in a subsequent clause, “which” would be inappropriate and introduce an ambiguity).RespectivelyThe word respectively is frequently misused by native and non-native English-speaking authorsalike, and, as with the other elements described above, its misuse can lead to confusion and ambiguities. It is often clearer not to use this term at all, but it can be useful to economise on words where there are two corresponding lists. For example, it is quite useful in the sentence “The latencies to withdrawal from a painful stimulus in control and transgenic mice were 3 s and 2 s, respectively”, meaning that control mice withdrew after 3 s and trans genic mice withdrew after 2 s. If describing something much shorter than “The latencies to withdrawal from a painful stimulus”, for example average weights, respectively is not necessary; “Control mice weighed 20±3 g and transgenic mice weighed 17±2 g” is better than “Control mice and transgenic mice weighed 20±3 g and 17±2 g, respectively”, which contains one additional word. Note that “respectively” can only be used to refer to two corresponding lists at one time, and cannot be used to refer to more. Thus, the sentence “The latencies to withdrawal from 5 g and 10 g painful stimuli in control and transgenic mice were 3 s and 2 s, respectively” is incorrect and impossible to understand.“The proportions of monocytes positive for CD163, CD7 and CD11a were 45%, 63% and 70%, respectively” (the “respectively” makes clear that the three percentages refer to each of the three markers in the same order).ComparisonsComparisons are frequently made in the results sections of papers, and it is especially important to compare “like with like”. One common error made by non-native authors is overlooking this simple rule and leaving the reader to make an assumption about what is being compared. At best, the language will appear unnatural but the meaning clear; at worst, the wrong meaning can be imparted. As an example, the sentence “Expression levels of p53 in smokers were compared with non-smokers” should actually be “Expression levels of p53 in smokers were compared with those in non-smokers”. Another frequent error with comparisons is the use of relativeterms (for example, higher, greater, more) without a reference. In the sentence “transgenic mice showed higher levels of cortisol” it is unclear what these levels were higher than; thus, a “than clause”, such as “than control mice”, is required. The reader might make this assumption automatically, but in some cases alternative inferences will be possible; the goal of accurate scientific writing has to be the removal of all assumption. Because comparisons of results are critical to their interpretation and, ultimately, their significance, it is critical that you convey to the reader exactly what is being compared. Finally, the word “between” should be used for comparisons of two findings, but “among” should be used for compa risons of three or more. 1. “The levels of ubiquitinated proteins were higher in patients than in control subjects” (the “than clause” provides a reference for the term “higher”).2. “The levels of ubiquitinated proteins in patients were higher than those in control subjects” (unlike the first example, where patients and controls are both on the same side of the comparing term, that is, they are both mentioned after “higher”, here, patients and controls appear either side of the comparing term; therefore, i t is necessary to add “than those” to compare like with like).3. “There was no significant difference in the levels of ubiquitinated proteins between patients and controls” (“between is appropriate here for a comparison of two groups).4. “There were no s ignificant differences in the levels of ubiquitinated proteins among AD patients, PD patients and controls” (among is appropriate for comparisons of more than two groups; note the change to the plural differences because more than one type of difference is possible with more than two groups).Protein and gene nomenclatureOne very common cause of confusion is use of the incorrect nomenclature to describe changes in the levels of genes, their mRNAs or the proteins that they encode. Constant changing fromdescribing gene expression levels to protein levels and back again can also add to the confusion, especially because the names are often the same. Therefore, it needs to be completely clear to the reader exactly what level you are talking about. Nomenclature differs among species, but generally gene names should be described in italics and protein names in normal font. Case (upper vs lower) is often used to distinguish between species: generally, for mouse, rat and chicken, gene names are spelt with an upper case first letter and the rest in lower case; for humans, primates and some domestic species, gene names are spelt with all capital letters. Descriptions of mRNAs generally use the gene name (for example, “levels of p53 mRNA”) or you can refer to the mRNA “for” a given protein (for example “levels of the mRNA for p53”). The word “expression” is usually used to describe gene expression and can induce confusion when used to describe protein and mRNA levels; in most cases referring to proteins the word “expression” can simply be replaced with the word “level” (or “levels”). Be aware of the correct nomenclature for your species of subject and ensure that everywhere you refer to a protein, gene or mRNA by name in the text it is completely clear which of those you are referring to.1. “Expression of the Igf1 gene was increased in our transgenic mice” (use if italics and the word “gene” ensure that no confusion is possible here).2. “The levels of IGF1 mRNA were elevated in our patient group” (correct nomenclature fo r human genes).3. “The serum IGF1 levels were elevated in the transgenic mice” (here, it is clear that the protein is being referred to; capitals are appropriate in this case, even though the species is mouse, because it is the correct nomenclature for the mouse protein).SummaryThese are just a few of the most common errors made by non-native English-speaking authorsin their scientific writing. There are of course many more that can‟t be dealt with here, but they all have the same result: a loss of clarity and/or introduction of ambiguity. If you apply the three “C”s when writing your next paper, with an awareness of some of the traps that can lead to ambiguities or a loss of clarity, you will automatically improve your chances of getting your study published. If you also focus on removing any repetition and redundancy, and apply attention to detail to ensure that your meaning is clearly conveyed in each sentence, you will increase them further. As a general rule, it is a good idea to keep sentences simple, using shorter expressions wherever possible, rather than long, complicated and confusing. The slogan for the Beijing Olympics was “One world, One dream”; when it comes to scientific writing you should think “One sentence, One idea”. The simplest solutio n is always the best.The cover letterDr. Daniel McGowan 论文写作系列第三讲The cover letter: your sales pitchDr. Daniel McGowan 将向大家展示如何写出吸引读者的“cover letter”。