A5. Recent Links and References - Biology

  1. Byres, E. Nature 456, 648-652 (2008)

  2. Pam Tangvoranuntakul, Pascal Gagneux, Sandra Diaz, Sandra Diaz, Ajit Varki, and Elaine Muchmore

    and Elaine Muchmore. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. PNAS 2003 100:12045-12050

  3. Cramer and Truhlar. Quantum Chemical Conformational Analysis of Glucose in Aqueous solutions. J. Am. Chem. Soc. 115, pg 5745 (1993)

Fisheries Biology Research Guide

Reference materials serve a variety of purposes and can sometimes be used effectively in searching the scientific literature. Reference materials can be used to:

  • define terminology--dictionaries
  • locate accepted knowledge and topical overviews--encyclopedias, handbooks and reviews
  • find factual information--directories and statistical compilations
  • identify research on specific topics--reviews and bibliographies
  • locate standard methods and procedures--handbooks and manuals

Following are reference materials of interest to the fisheries scientist and manager. They are organized by category of reference material rather than subject. Resources marked with a are key resources or databases.

Table of Contents

Your application's Research Plan has two sections:

  1. Specific Aims—a one-page statement of your objectives for the project.
  2. Research Strategy—a description of the rationale for your research and your experiments in 12 pages for an R01.

In your Specific Aims, you note the significance and innovation of your research then list your two to three concrete objectives, your aims.

Your Research Strategy is the nuts and bolts of your application, where you describe your research rationale and the experiments you will conduct to accomplish each aim. Though how you organize it is largely up to you, NIH expects you to follow these guidelines.

  • Organize using bold headers or an outline or numbering system—or both—that you use consistently throughout.
  • Start each section with the appropriate header: Significance, Innovation, or Approach.
  • Organize the Approach section around your Specific Aims.

Format of Your Research Plan

To write the Research Plan, you don't need the application forms. Write the text in your word processor, turn it into a PDF file, and upload it into the application form when it's final.

Because NIH may return your application if it doesn't meet all requirements, be sure to follow the rules for font, page limits, and more. Read the instructions at NIH’s Format Attachments.

For an R01, the Research Strategy can be up to 12 pages, plus one page for Specific Aims. Don't pad other sections with information that belongs in the Research Plan. NIH is on the lookout and may return your application to you if you try to evade page limits.

Follow Examples

As you read this page, look at our Sample Applications and More to see some of the different strategies successful PIs use to create an outstanding Research Plan.

Keeping It All In Sync

Writing in a logical sequence will save you time.

Information you put in the Research Plan affects just about every other application part. You'll need to keep everything in sync as your plans evolve during the writing phase.

It's best to consider your writing as an iterative process. As you develop and finalize your experiments, you will go back and check other parts of the application to make sure everything is in sync: the "who, what, when, where, and how (much money)" as well as look again at the scope of your plans.

In that vein, writing in a logical sequence is a good approach that will save you time. We suggest proceeding in the following order:

  1. Create a provisional title.
  2. Write a draft of your Specific Aims.
  3. Write your Research Strategy.
    • Start with your Significance and Innovation sections.
    • Then draft the Approach section considering the personnel and skills you'll need for each step.
  4. Evaluate your Specific Aims and methods in light of your expected budget (for a new PI, it should be modest, probably under the $250,000 for NIH's modular budget).
  5. As you design experiments, reevaluate your hypothesis, aims, and title to make sure they still reflect your plans.
  6. Prepare your Abstract (a summary of your Specific Aims).
  7. Complete the other forms.

Even the smaller sections of your application need to be well-organized and readable so reviewers can readily grasp the information. If writing is not your forte, get help.

To view writing strategies for successful applications, see our Sample Applications and More. There are many ways to create a great application, so explore your options.

Annexin A5 Is Not Essential for Skeletal Development

FIG. 1 . (A) Generation of annexin A5-deficient mice. The structures of the wild-type allele of the Anxa5 gene in the 129/SvJ (E14) and C57BL/6 mouse strains, the targeting vector, and the disrupted allele are shown with numbered exons (vertical bars) and introns. The presence of MuERV is shown. LacZ and neomycin (Neo) cassettes are marked in the targeting construct, and regions of homology are indicated (grey lines). The sizes of the EcoRV fragments, detected by a probe specific for exon 6 (asterisks), are 7 and 12.5 kbp for the wild-type alleles of C57BL/6 and 129/SvJ (E14) mice as well as 8 kbp for the disrupted allele, respectively. (B) Southern blot analysis of offspring from heterozygous intercrosses digested with EcoRV and hybridized with the probe exon 6. Mut, mutant Wt, wild type. (C) PCR analysis of isolated DNA results in fragments of 301 bp for the wild type and of 449 bp for the disrupted allele. FIG. 2 . Expression of annexin A2, A5, A6, and A7 in organs from wild-type (+/+) and annexin A5-deficient (−/−) mice. Protein samples of tissue lysates (20 μg of total protein per lane) from the liver, lung, spleen, and heart were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of annexins A2, A5, A6, and A7 was detected by immunoblotting with specific antibodies. To avoid contamination with proteins from neighboring lanes, an empty lane separated the wild-type and annexin A5-deficient lanes. FIG. 3 . Cartilage and bone develop normally in newborns lacking annexin A5. The skeletons from newborn littermates of different genotypes (+/+, +/−, and −/−) were stained with alcian blue and alizarin red, detecting cartilage and bony structures, respectively. FIG. 4 . Expression of annexin A5 in the tibia of wild-type (+/+) (A, C, E, and G) and annexin A5-deficient (−/−) (B, D, and H) mice. (A and B) Phase contrast image of sections from the tibia. (C and D) Detection of annexin A5 protein by immunohistochemistry. (E) Higher magnification of panel C. (F) Immunostaining with the secondary antibody as negative control. (G and H) Parallel staining for β-galactosidase activity by X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) substrate (blue) and for calcium deposits by alizarin red. Bars, 250 μm. FIG. 5 . In vitro calcification of isolated chondrocytes from wild-type (A, C, E, and G) and annexin A5-deficient (B, D, F, and H) mice. (A and B) Chondrocytes were induced for 8 days in medium supplemented with 10 mM glycerophosphate, 10 mM CaCl2, and 50 μg of ascorbate/ml stained for mineral deposits with 0.5% alizarin red and cleared with 2% potassium hydroxide. (C and D) Higher magnifications of panels A and B, respectively, are shown. Bar, 250 μm. (E and F) Single cell clusters at higher magnifications of panels C and D, respectively, are shown. Bar, 50 μm. (G and H) Chondrocytes cultured in medium without induction are shown.

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Table of Paper Sizes From 4A0 to A10

SizeWidth x Height (mm)Width x Height (in)
4A01682 x 2378 mm66.2 x 93.6 in
2A01189 x 1682 mm46.8 x 66.2 in
A0841 x 1189 mm33.1 x 46.8 in
A1594 x 841 mm23.4 x 33.1 in
A2420 x 594 mm16.5 x 23.4 in
A3297 x 420 mm11.7 x 16.5 in
A4210 x 297 mm8.3 x 11.7 in
A5148 x 210 mm5.8 x 8.3 in
A6105 x 148 mm4.1 x 5.8 in
A774 x 105 mm2.9 x 4.1 in
A852 x 74 mm2.0 x 2.9 in
A937 x 52 mm1.5 x 2.0 in
A1026 x 37 mm1.0 x 1.5 in

To obtain paper sizes in centimetres, convert mm values to cm by dividing by 10 and in feet by dividing inch values by 12. More units here and sizes in pixels here.

Annexin A5: an imaging biomarker of cardiovascular risk

Apoptosis, a form of programmed cell death (PCD), plays an important role in the initiation and progression of a number of cardiovascular disease, such as heart failure, myocardial infarction, and atherosclerosis. One of the most prominent characteristics of apoptosis is the externalisation of phosphatidylserine (PS), a plasma cell membrane phospholipid, which in healthy cells only is present on the inner leaflet of the plasma cell membrane. Annexin A5, a 35 kD plasma protein, has strong affinity for PS in the nano-molar range. Through the coupling of Annexin A5 to contrast agents, visualization of apoptotic cell death in vivo in animal models and in patients has become feasible. These imaging studies have provided novel insight into the extent and kinetics of apoptosis in cardiovascular disease. Furthermore, Annexin A5 imaging has proven to be a suitable imaging biomarker for the evaluation of cell death modifying compounds and plaque stabilizing strategies. Recent insight in PS biology has shown that PS externalisation not only occurs in apoptosis, but is also observed in activated macrophages and stressed cells. In addition, it has been shown that Annexin A5 not only binds to exteriorized PS, but is also internalized through an Annexin A5 specific mechanism. These latter findings indicate that Annexin A5 imaging is not exclusively valuable for apoptosis detection, but can also be used to visualize inflammation and cell stress. This will open novel opportunities for imaging and drug delivery strategies. In this review we will discuss the introduction of Annexin A5 in preclinical and clinical imaging studies and provide an outlook on novel opportunities of Annexin A5 based targeting of PS.

This is a preview of subscription content, access via your institution.

Imidazoquinoxaline derivative EAPB0503: A promising drug targeting mutant nucleophosmin 1 in acute myeloid leukemia

Background: Nucleophosmin 1 (NPM1) is a nucleocytoplasmic shuttling protein mainly localized in the nucleolus. NPM1 is frequently mutated in acute myeloid leukemia (AML). NPM1c oligomerizes with wild-type nucleophosmin 1 (wt-NPM1), and this leads to its continuous cytoplasmic delocalization and contributes to leukemogenesis. Recent studies have shown that Cytoplasmic NPM1 (NPM1c) degradation leads to growth arrest and apoptosis of NPM1c AML cells and corrects wt-NPM1 normal nucleolar localization.

Methods: AML cells expressing wt-NPM1 or NPM1c or transfected with wt-NPM1 or NPM1c as well as wt-NPM1 and NPM1c AML xenograft mice were used. Cell growth was assessed with trypan blue or a CellTiter 96 proliferation kit. The cell cycle was studied with a propidium iodide (PI) assay. Caspase-mediated intrinsic apoptosis was assessed with annexin V/PI, the mitochondrial membrane potential, and poly(adenosine diphosphate ribose) polymerase cleavage. The expression of NPM1, p53, phosphorylated p53, and p21 was analyzed via immunoblotting. Localization was performed with confocal microscopy. The leukemia burden was evaluated by flow cytometry with an anti-human CD45 antibody.

Results: The imidazoquinoxaline 1-(3-methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (EAPB0503) induced selective proteasome-mediated degradation of NPM1c, restored wt-NPM1 nucleolar localization in NPM1c AML cells, and thus yielded selective growth arrest and apoptosis. Introducing NPM1c to cells normally harboring wt-NPM1 sensitized them to EAPB0503 and led to their growth arrest. Moreover, EAPB0503 selectively reduced the leukemia burden in NPM1c AML xenograft mice.

Conclusions: These findings further reinforce the idea of targeting the NPM1c oncoprotein to eradicate leukemic cells and warrant a broader preclinical evaluation and then a clinical evaluation of this promising drug. Cancer 2017123:1662-1673. © 2017 American Cancer Society.

Keywords: 1-(3-methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (EAPB0503) acute myeloid leukemia apoptosis nucleophosmin 1 xenograft mice.

Brain Science A5 Hardcover

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In this study, beyond providing up-to-date estimates on the average values of the number of cells, we aimed to give representative uncertainty ranges and the variation across population segments. This is based on comparing independent studies and the variation observed within studies.

The biggest knowledge gap we find is how realistic is the usage of the measured fecal bacteria density to represent also the average bacteria density in the colon. There is an inevitable gradient in bacteria concentration along the colon itself, from the low concentrations transiting from the ileum to the cecum of about 10 8 bacteria/g to

10 11 bacteria/g measured in stool. The change in bacteria concentration arises from several factors, including water absorption that concentrates the bacteria in the colon, as well as from bacteria growth during the 1𠄲 day transit time and the shedding of bacteria from the mucosal surface. In some respects, the estimate we performed of multiplying observed fecal bacteria density with colon content volume can be considered an upper limit. More information on the relation between the actual densities of bacteria throughout the colon and those densities measured in feces will be a big step forward in improving the estimates of this study. Another element of uncertainty is the limited information on the volume of the colon content across individuals and conditions. These knowledge gaps indicate that there might be systematic errors beyond what we could account for in the uncertainty ranges we report.

In analyzing various population segments, our paper is clearly limited in scope. We touched on the obese, neonate, and elderly as well as the effect of gender but have not dealt with many other segments of interest, such as individuals in the course of antibiotic treatment or bowel preparation for colonoscopy, people with infections, chronic diseases of the GI tract, etc.

While we analyzed cell numbers, many researchers are interested in the number of genes as a reflection, for example, of the diversity of the microbiome metabolic capabilities. In order to properly estimate by what factor the genes in the bacteria we harbor outnumber our own twenty thousand genes, the very delicate question of what should be considered different genes must be properly defined, which is beyond the scope of this study.

We note in passing that the number of endosymbiotic bacteria that we harbor in the form of mitochondria probably outnumbers the body bacteria several fold. This can be appreciated by noting that most cell types (though not red blood cells) contain hundreds (or more) of mitochondria per cell [48].

Should we care about the absolute number of human cells in the body or the ratio of bacterial to human cells? Updating the ratio of bacteria to human cells from 10:1 or 100:1 to closer to 1:1 does not take away from the biological importance of the microbiota. Yet, we are convinced that a number widely stated should be based on the best available data, serving to keep the quantitative biological discourse rigorous. The study of absolute numbers is also of relevance for specific biological questions. For example, a recent study showed how knowing the number of cells in different tissues can be an important indicator in understanding variation in cancer risk among tissues [49]. Other applications refer to the dynamic processes of development and mutation accumulation. Finally, the type of numeric focus exercised here reveals and attracts attention to knowledge gaps such as the bacterial population densities in the proximal colon and how well are they represented by current analysis methods. We thus became aware through this study of promising steps forward in fulfilling the Delphic maxim of ‘‘know thyself” from a quantitative perspective.

Watch the video: RNA-Interferenz - Mechanismus zum Abschalten von Genen - Biologie, Genregulation, Oberstufe (January 2022).