DARWIN Y LA EVOLUCIÓN BIOLÓGICA

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DARWIN Y LA EVOLUCIÓN BIOLÓGICA

Charles Robert Darwin (12 de febrero de 1809 — 19 de abril de 1882) fue un naturalista inglés que postuló que todas las especies de seres vivos han evolucio…

Charles Robert Darwin (12 de febrero de 1809 — 19 de abril de 1882) fue un naturalista inglés que postuló que todas las especies de seres vivos han evolucio…

Evolução, Charles Darwin e Seleção Natural Aula Grátis de Biologia – Teoria da Evolução e Darwinismo

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Evolução, Charles Darwin e Seleção Natural Aula Grátis de Biologia – Teoria da Evolução e Darwinismo

Charles Darwin foi um dos homens que teve maior influência sobre o pensamento científico do século passado ao publicar suas idéias sobre evolução e seleção n…

Charles Darwin foi um dos homens que teve maior influência sobre o pensamento científico do século passado ao publicar suas idéias sobre evolução e seleção n…

Darwin’s Struggle: The Evolution Of The Origin Of Species (BBC)

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Darwin’s Struggle: The Evolution Of The Origin Of Species (BBC)

Documentary telling the little-known story of how Darwin came to write his great masterpiece, On the Origin of Species, a book which explains the wonderful v…

Documentary telling the little-known story of how Darwin came to write his great masterpiece, On the Origin of Species, a book which explains the wonderful v…

Evolution – What Darwin Never Knew – NOVA PBS Documentary

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Evolution – What Darwin Never Knew – NOVA PBS Documentary

DOWNLOAD BOOK ( pdf ): http://www.mediafire.com/?39l9wwiurjns48y READ ON-LINE: http://archive.org/stream/originofspecies00darwuoft#page/n0/mode/2up.

DOWNLOAD BOOK ( pdf ): http://www.mediafire.com/?39l9wwiurjns48y READ ON-LINE: http://archive.org/stream/originofspecies00darwuoft#page/n0/mode/2up.

Evolution – Part 1 of 7 – Darwin’s Dangerous Idea (PBS Documentary)[HD 720p]

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Evolution – Part 1 of 7 – Darwin’s Dangerous Idea (PBS Documentary)[HD 720p]

Darwin’s Book : “Origin of Species” Download ( pdf ): http://www.mediafire.com/?39l9wwiurjns48y Read: http://archive.org/stream/originofspecies00darwuoft#pag…

Darwin’s Book : “Origin of Species” Download ( pdf ): http://www.mediafire.com/?39l9wwiurjns48y Read: http://archive.org/stream/originofspecies00darwuoft#pag…

The Genius Of Charles Darwin – Part 1

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The Genius Of Charles Darwin – Part 1

The Genius Of Charles Darwin – Part 1: The man who has surpassed all generations in critical thinking

Join our facebook debate group; http://www.facebook.com/CreationEvolutionDebate Join me on facebook http://www.facebook.com/pages/TheScienceFoundation/277697…

collection of human evolution fossils

The Genius Of Charles Darwin – Part 1: The man who has surpassed all generations in critical thinking Join our facebook debate group; http://www.facebook.com/CreationEvolutionDebate Join me on facebook http://www.facebook.com/pages/TheScienceFoundation/277697…

Charles Darwin ( The Man That Changed The World )

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Charles Darwin ( The Man That Changed The World )

Text: In 1859, one man changed the world forever. He was Charles Darwin. Part1: The Breakthrough He was born in Shrewsbury, Shropshire, England on 12 Februar…

Text: In 1859, one man changed the world forever. He was Charles Darwin. Part1: The Breakthrough He was born in Shrewsbury, Shropshire, England on 12 Februar…

Erythrocytes, Red Blood Cell Lysis, Hemolytic Assay in Whole Blood

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Hemolytic Assay in Whole Blood: A relevant assay that is reflective of the in vivo cytotoxic potential of a drug [e.g., antimicrobial peptides (AMPs)] when administered systemically

Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood

 Background:

 Hemolytic assay in phosphate buffered saline (PBS) is routinely used to examine toxic effects of a particular drug on human erythrocytes (1). This assay is widely accepted for determining potential toxic effects on red blood cells, particularly in the context of structure-function studies. While informative, the hemolytic property of a drug [e.g., antimicrobial peptides (AMPs)] in PBS is not a realistic measure of its toxic potential in vivo. As we have demonstrated (2), the antimicrobial selectivity of antimicrobial peptides in serum or whole blood is different from that determined in PBS. Both antimicrobial and hemolytic effects in blood substantially decrease. In fact, it was known for years that AMPs are inactivated in serum; their antimicrobial activity and their hemolytic activity are dampened in blood when compared to PBS. Hence, as a general principle, both antimicrobial activity and host toxicity should be examined in the context of an environment that is reflective of the ultimate application of the drug.

It is because of the observation of this serum or blood effect that the hemolytic assay in whole blood was developed. Interestingly, this assay is not very different from the standard assay in PBS, except for one or two steps. However, here I will describe the assay completely.

 Materials:
  • Heparinized blood or uncoagulated blood
  • Red blood cell lysis buffer or just deionized water (recommended)
  • A U-bottom 96-well plate
  • 1.5 ml eppendorf tubes
  • A flat-bottom 96-well plate
  • A 98-well plate reader
  • The drug you are studying
Methods:
  1. Treat the blood with your drug(s) of interest
  2. Prepare the drug in a U-bottom 96-well plate using serial dilutions from an 8x maximum test concentration (80, 40, 20 μM, etc.)
  3. Be sure that the drug is prepared in 50 μL volume prior to serial dilutions. After the dilutions, there will be 25μL in each well at 8x the ideal test concentrations. Please be sure that there are 2 wells in each column with no drug (only PBS)
  4. To each well, add 175μL of uncoagulated blood and incubate for at least one hour at 37⁰C. Please note that, in standard red blood cells lysis in PBS, the RBCs are isolated from the blood and resuspended in PBS at 5-10% prior to drug exposure. Here, we simply use the erythrocytes in their natural environment to perform the assay.
  5. Prepare your standard samples
    1. Using 8 eppendorf tubes, add 450 to 500 uL of dH20 (or RBC lysis buffer)
    2. Transfer 0, 5 (10% lysis), 10, 20, 30, 40 (80% lysis), and 50 μL of whole blood from a well-mixed sample to each tube up to a volume of 500μL. Incubate at room temperature for at least 15 minutes and be sure to vortex before use.
    3. Add 200μL of standard to one column of flat-bottom 96-well plate for future spectrophotometric analysis with your test samples
  6. Prepare your test samples for measurement of hemoglobin release
    1. Centrifuge your test plate (the one with the blood treated with the drug) at 500G for 5-10 minutes. Your supernatants contain the red hemoglobin if your drug is hemolytic.
    2. In the flat-bottom plate, add 178 uL of dH20. This step is critical, and here is why: The concentration of RBC in your standard is 0-10% (you could argue 5% since RBC is about 50% of the blood volume).  Remember the blood was diluted up to 1:10. Then when the drug lyses the RBCs (hypothetically), the hemoglobin released from your sample was only diluted about 9:10 (175/200). These samples are up to 9x more concentrated compared to your standard. This means that you have to dilute your samples 1/9 so that the concentrations of hemoglobin released in your test samples are comparable to the blood or RBC concentrations used for your standards. If this is not clear, please send me a question.
    3. Carefully remove 22μL from the top layer of each of your test wells and add it to 178μL in corresponding wells of the flat-bottom plate already containing your standards (feel free to read all samples in duplicates)
    4. Place the plate in a plate reader and read at a wavelength between 550 and 570nm
  7. Data analysis
    1. Standard curve: Below is an example of an actual standard curve I generated from an RBC lysis assay in  whole blood:

 

hemolytic assay: rbc lysis assay standard curve

 

STD RBC (% lysis) ABS570
100 0.5905
80 0.5145
60 0.3855
40 0.264
20 0.167
10 0.0895
0 0.037

All you have to do now is to replace the ABS570 for each of your test sample in the derived equation to find the percent hemolysis.

% hemolysis (test) = [(Abs570 -0.0416)/0.0057];
(some  use ABS550 as well)

I hope I did not leave any step unclear. If I did, please send me a message from the comment box.

Best of luck with your experiment!

 Selected References

1.  Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens.

Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Mietzner TA, Montelaro RC.

Antimicrob Agents Chemother. 2013 Jun;57(6):2511-21. doi: 10.1128/AAC.02218-12. Epub 2013 Mar 18.

PMID: 23507278 [PubMed - indexed for MEDLINE] Free PMC Article

2. Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

Deslouches B, Islam K, Craigo JK, Paranjape SM, Montelaro RC, Mietzner TA.

Antimicrob Agents Chemother. 2005 Aug;49(8):3208-16.

PMID: 16048927 [PubMed - indexed for MEDLINE] Free PMC Article
Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood
hemolytic assay: rbc lysis standard curve

Hemolytic Assay in Whole Blood: A relevant assay that is reflective of the in vivo cytotoxic potential of a drug [e.g., antimicrobial peptides (AMPs)] when administered systemically Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood  Background:  Hemolytic assay in phosphate buffered saline (PBS) is routinely used More Info »

Luloot Academy mission: connect teachers, students, scholars

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Luloot Academy, the mission: to connect students, teachers, and scholars in all academic disciplines to facilitate sharing of knowledge

To share or to learn how to seek the naked truth

Founder of Luloot Academy Berthony Deslouches, MD, PhDLuloot Academy is an online academy with the mission to make knowledge accessible to all and particularly (when possible) the least fortunate. The academy is designed to be a community effort involving all educators such as teachers, scientists, literary critics, and other scholars in addition to students.

How can you use this website efficiently?

1. The first thing you need to do is to sign-up for an account to start learning or contributing

2. You can post the following types of materials

  • Book chapter-style reviews:
  • Expert opinions or analyses: you may have an interesting point of view based on existing facts. We would love you to share that on Luloot Academy.
  • It is recommended that our staff as well as current users on Luloot Academy review your posts and propose edits intended to make your articles read better.
  • Scientific protocol submission is highly encouraged!
  • Lesson or lecture notes: teachers may post their lecture notes or even lesson plans online.
  • Video lectures or other types of educational videos
  • Importantly, your membership entitles you to a website that looks just like this one or it can be custom-designed for you. All websites associated with the academy should be consistent with its mission to share knowledge or to promote professional development. This means that your website can be used for self-promotion as a professional, student, teacher, or scholar with your professional achievements or video profile. In addition, a website can just be about a specific topic in any academic discipline! We will design your website at yoursite.luloot.com. Once you sign up, just send us a support ticket with a description of desirable features.
  • Your website will be listed in our directory

Future plan: Luloot Academy has some exciting new features that may become within the next 2-3 years

  • An academic network to better connect members of the academic community with all standard features that facilitate free sharing of information such as
    • Chat (currently available), video chat, and video conferencing
    • Discussion forums with questions and answers (currently available)
    • Online curriculum vitae (CV) or resume (currently available using the professional or business listing module)
    • Business and academic job posting
    • Online debate module for discussion of opinions or research articles on any topics from politics to religion or science (currently available using the forum or Q&A modules)
    • Exam review module consisting of multiple choice questions that mimic standardized exams with review answers at the end of each exam. The option for Links to videos  or images to better illustrate specific concepts may be provided
  • Users’ achievements will be highlighted on Luloot Academy. Such achievements include, but are not limited to, professional promotion, grant awards, and academic honors.
  • The virtual dynamic classroom:

    • This is a the ultimate project that will take some time to execute. The goal of the virtual dynamic classroom is for Luloot Academy to offer something uniquely lulootic! The virtual dynamic classroom is the transformation of traditional, lecture-like teaching to a demonstrative, interactive teaching or sharing of knowledge. How it will work is that Luloot Academy will create movie-like educational videos displaying 3D models that look realistically human.
    • To give you an idea about this virtual dynamic classroom:  human-like 3D models will be displayed inside  or outside a classroom environment in a very interactive manner that is designed to demonstrate or to teach a concept in the context of a story. It is like a movie except that it is designed to teach. Of course, this will be a costly program that will take time to implement. I want Luloot Academy to be freely accessible to all, but the reality is human activities cost money. Thus,  Luloot Academy unfortunately requires a monthly fee of $9.99 for permanent access (which includes your professional website design hosted on our network). Nevertheless, I hope that you will still find Luloot Academy an excellent platform conducive to free sharing of standard information for all!
  • What are the rules?
    • (i) No foul language or language of violence and hatred is acceptable on Luloot Academy. (ii) All materials must comply to the grammatical standard of the English language. (iii) This is a platform for learning and professional networking. All accounts should be authentic. That is you must identify yourself on Luloot Academy at least with your real name and professional title (if any).
    • If you observe these three rules (as I do), you will be alright!
    • A special thanks to you for visiting Luloot Academy!
Luloot Academy mission: to share and learn how to seek the truth

Luloot Academy, the mission: to connect students, teachers, and scholars in all academic disciplines to facilitate sharing of knowledge To share or to learn how to seek the naked truth Luloot Academy is an online academy with the mission to make knowledge accessible to all and particularly (when possible) the least fortunate. The academy is designed More Info »