Q&A

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  • Patrick Thomas Knott added an answer in Posture:
    Who is currently working with 4D analysis of posture?

    I want to work with 4D analysis of posture, but we bought it recently and we don't know how work with it. How do we analyze posture and how collect data?

    Patrick Thomas Knott · Rosalind Franklin University of Medicine and Science

    I have done 4D posture measurement.  Please provide more info about your question.

  • What is an effect size in ordinal logistic regression?
    I am not sure whether the percent increase/decrease in odd ratios of binary logistic regression can be used as a measure of effect size in ordinal logistic regression too. I would greatly appreciate some pointers towards the estimation of effect size in ordinal logistic regression.
    Nabin Baral · University of Washington Seattle

    Thank you.

  • Thelma Vance added an answer in 2D & 3D:
    How can we reduce n dimensional features in mining technique?

    Normally we can do the operation using 2D or 3D datasets in clustering technique, which can be calculated using normal distance calculating formula but used on higher dimension, I will get wrong result. Is there any proximity measure technique(distance formula) used in higher dimensional datasets?

    Thelma Vance · Northcentral University

    What happens if the dimension is 'curved space' or a twisted manifold?

  • Hao Chen added an answer in Transgenic Mice:
    Has anyone ever conducted electrophysiological test like LTP test on 3xTg mice?

    According to JAX, it is not recommended to use male 3xTg mice as tested subjects because of the report that male transgenic mice may not exhibit the phenotypic traits originally described. But for eletrophysiological test, male mice are usually preferred due to little estrogen effect. So is it a good method to test their LTP? Thanks a lot!

    Hao Chen · South University of Science and Technology of China

    Luke Searcy: Thanks! I have skimmed the paper and you have done great job! And I noticed all mice you used were female and wonder whether their estrogen level can affect the result. Could you tell me why you chose female then? Considering electrophysiological study was conducted over their 14 months, is the estrogen effect limited on postmenopausal mice? Or estrogen effect are actually not a big concern because their cycles can be synchronized via group housing? 

    Thank others also! Previous works are helpful for us!

  • Julie Lynn Horton added an answer in OLIGO:
    I would like to know If I have pCRISPR and pCas9, what is the precise sequence of the oligos that is complementary to the undesirable gene?

    I knew already the oligos length is about 30 bp but I do not know of which part of the undesirable gene in the bacterial genome?

    Julie Lynn Horton · Sanford-Burnham Medical Research Institute

    If I understand your question correctly you are trying to design an oligo sequence to be your guide RNA. If that is the case, you want to meet certain criteria when designing the oligo. You want to 1)make sure the sequence is present in all transcript variants. 2) Make sure the sequence is followed by an NGG (PAM) motif, and 3) Make sure the sequence has as few off-targets as possible. 

  • I go through BLAST for the high similarities... Now how to produce the best phylogenetic tree?
    I have TSM 16S ribosomal RNA gene
    Saher Shahid · University of the Punjab

    when i blast a protein sequnece, it shows homology with many putative proteins too. Should i skip those sequences? 

  • Ariananda Hariadi added an answer in Trizol:
    How do you dissolve DNA pellet in TE Buffer?

    I have extracted gDNA from mononuclear cells using TRIZOL reagent, but it doesn't get dissolved in TE buffer. Why is that so? And is it possible to dissolve it in TE buffer?

    Please kindly share your experience and method to dissolve extracted DNA in TE buffer. Thanks!

    Ariananda Hariadi · Dharmais Hospital National Cancer Center

    I had decided to incubate my sample in 60C for 10 minutes, and it worked successfully. There was still a gooey clot remain but after I dissolve twice the volume and incubate it for another 10 minutes it turned out just fine and the yield was also quite high (around 500 ng/ul.) Thanks for all your answers! :)

  • Will mycoplasma infection have any effect on my lentiviral transduction?

    Hi, I have started to optimize my cell line for Lentiviral transduction and I found that my cells were positive for mycoplasma contamination again although this was a new batch of cells. I will be starting to treat the cells with Plasmocin in a day or two. However, since I have already started my optimization of Lentiviral transduction for shRNAs, I would like to know if my optimized values will be affected by mycoplasma infection or not? 

    Ulrike Jung · City of Hope National Medical Center

    I would absolutely discard anything that has been infected with Mycoplasma because the infection of course does affect your cell gene expression pattern. You might treat them if they are not replaceable but it is likely your results downstream will differ from cells that were never infected. In that case reproducibility of your experiments is at risk and retraction of your results if published can happen in the worst case.

    Mycoplasma contamination can be transferred in liquid nitrogen from what I read, so if your vials are all contaminated, it is either from the initial batch of cells or maybe through the liquid nitrogen tank. I have had Mycoplasma once in my career and it was through a special cell line from another lab. Clean working and very strict safety precautions allowed me thankfully to avoid contaminating anything else and we got rid of the cells quickly. I would urge you to do the same if you can afford it at all!

    For the question about transfection: do you mean transfection for lentiviral packaging or lentiviral transduction? Colleagues have told me it affects transfection of 293T clearly so if they have trouble with that always immediately check for mycoplasma first. As I said I have no experience myself but there is enough literature out there to support that notion.

    https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/ for example.

  • Why is financial inclusion in India a far-off destination, despite mandatory policy regimes & deadlines?

    Myriad policy prescriptions and awareness creation have passed. But financial inclusion in India is still a distant dream. I'm doubtful about the viability of idealistic recommendations of experts.

    Haradhan Kumar Mohajan · Premier University

    India is large country with densely populated and illiteracy rate is also very high. Due to burden of most unskilled human capital the country need time to be financially developed.

       
  • Maria Amaral added an answer in Gel Electrophoresis:
    Smear on agarose gel from QIAGEN extracted plant samples
    Would anyone know if it is common to observe these types of smears on an agarose gel after extracting DNA using a QIAGEN DNeasy 96 kit and running 500 ng of DNA in a 1% gel at 70 volts for 3 hours inside a 4 Celsius fridge (also tried at room temperature)?

    I'm planning on sending these samples for Genotyping-by-Sequencing but the requirements for such procedure include no smearing on agarose gel neither discrete low molecular weight bands, which I think I have. Any input will be appreciated.
    Maria Amaral · Université de Fribourg

    Hi Jerry,

    I have just joined this Rgate because of the problem you share here.

    I am working with DNA from a variety of tropical plant species and for some, I get gel pictures exactly like yours. The smear may depend on some DNA degradation that has already occurred in your samples, or that is happening during extraction. What kind of samples did you worked with?

    For the bands, I search on the net and I got 2 possible answer, so far, for this: lipopolysacharides with bind to ethidium bromide (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208550/ ), and / or DNA fragmentation. (file:///C:/Users/Joao/Downloads/JCT20110500005_97044297.pdf ).

    I tried to isolate the high molecular band fraction from gels, but this will made me loose too much DNA. I may just assume the risk and submit the samples as are, but I would help me a lot to know what decisions you made and if the results were ok.

    I hope you see this post and get back to me.

    Cheers

  • Zuzanna Michalak asked a question in Mining:
    Why the Western blot for SCN1a (+200 kD) is not working anymore with 5M Urea buffer brain proteins extract?

    I am running western blot to detect transmembrane protein of >200 kDa (SCN1a). I extracted my Proteins with 5M Urea Buffer, and I keep them at 4*C. I run Western using Tris-Acetate gradient gels 3-8 % with Tris acetate running buffer (Invitrogen). I prepare my samples in Laemli buffer (BioRad) or Sample buffer (from Invitrogen) with BetaMercapto or LDS as reducing agents. I do not boil the samples. I use wet transfer for 2h at 30V.

    The westerns worked very well, and suddenly from one day to another it stooped. First progressively my high MW band that before were very strong after 5 min (in the cassette) started to fade, then my Low MW, and by the end using the same samples I was unable to detect any band after few hours!

    Please give me any possibilities what the problem is. I tried to resolve the technical problem by changing the lot of gels, running buffers, Laemli, secondary and primary antibodies, electrophoresis and transfer systems...

    And the last test my friend performed for me using hers and mine samples, and all samples looked ok on PonceauS but the blot worked only on her samples - meaning there is something wrong with mine!

    I need to add that these samples are VERY important and impossible to reproduce (as it is a human material).

    I will be very thankful for any advices!

    Regards,

    Zuzanna

  • Mamatha M Pillai added an answer in Formazans:
    How can I dissolve the formazan crystals of cells which have been grown on a scaffold?

    I am working on differentiation of MSCs into osteoblasts by using electrospun scaffolds. I've used MTT assay to evaluate the toxicity of scaffolds and the proliferation of the cells. But i could not dissolve the formazan crystals of cells by DMSO because they surrounded by the nanofibers of scaffold. moreover I added the solvent of scaffold plus DMSO to dissolve the crystals but it was not successful. So i wonder if you could help me and give me a suggestion!

    Mamatha M Pillai · PSG Institute of Advanced Studies

    you can use DMSO to dissolve crystals

  • Kumara Ekanayake added an answer in Maxillary Sinus:
    What is the best way to manage keratocystic odontogenic tumours encroaching the maxillary sinus?

    Invasion of skull base will be difficult to manage.

    Kumara Ekanayake · Trinity College Dublin

    Thank you Lakshmanan

  • Mohammad Javad Razavi added an answer in Fatigue:
    Is there any fixture to do biaxial fatigue test using the uniaxial fatigue test machine?

    Dear researchers, I want to do some biaxial fatigue testing using my uniaxial fatigue machine! Does any body know some fixtures to invert the uniaxial loading to biaxial loading (for cruciform specimen)?

    Mohammad Javad Razavi · Iran University of Science and Technology

    Thank you all!

  • Ivan Madan added an answer in Phosphorescence:
    What the difference between singlet and trilet in Pi-electron state of a scintillation material?

    and difference b/w Fluorescence and Phosphorescence?

    Ivan Madan · Jožef Stefan Institute

    Difference is in a total spin of an exciton. Let's assume you excite a Pi-electron system which in it's ground state has even number of electrons with total spin 0. If you excite electron by a photon allowed transition would be those which conserve the spin. Now you are in an excited singlet state (S is zero) and photon emission is governed by the same selection rules as it was for absorption previously, emission is highly probable so you have fast (+-~ns) fluorescene. If in excited state spin flip you'll have a triplet state with typically lower energy. Optical transition is prohibited and lifetime is several orders of magnitude larger, this is called phosphorescence.

    For reading I would recommend Principles of Fluorescence Spectroscopy by
    J. Lakowicz.

  • Paul Sanders added an answer in E-Learning:
    What is your institution's policy on size of a cohort?
    Do you agree with the policy? If not, what are your concerns and would you suggest? I'm looking for any research, suggestions, or views on the size of a cohort specifically for online courses. This can be for undergraduate as well as graduate programs.
    Paul Sanders · United States Army Command and General Staff College

    Sixteen is the current cohort size for our resident course program and for online instructors like me we try to start the course with 16-18 students per cohort to allow for any initial attrition.  Currently, I facilitate two cohorts through our 52 week Advanced Operations Course.  In my opinion the ideal situation would be to facilitate just one cohort at a time to allow for more timely and robust feedback to each learner.  With 2 cohorts, certain short cuts must be taken regarding quality feedback. 

  • Louis Brassard added an answer in Qualia:
    What role has anticipation in consciousness?

    Philosophers says the brain is an anticipation machine. I miss this statement in the present discussions of consciousness on RG. Is there any interrelation of consciousness, Qualia and anticipation?

    Barbara,


    The Gestaltists and MP have done a lot for demonstrating that there is not a separation of labor between cognition and perception , that perception is intrinsically cognitive, that perception is always about type of things and that the senses is not dum and in needed of intelligence to make senses. In fact, intelligence not lead by the intelligence of the senses (Arnheim) get lost in the vap of discourses.
    Thank you for the David Huron’reference. I am trying to developed a model of humans based on a new perspective about what a body is. In that perspective, a body is what is integrated into the control or participation of a nervous system. So body can be extended and can be coupled and merged. In that conception, the evolution of mammalian type of body up to the primate lead to a new type of control of the body which allow their polymorphic extension and merging into all forms of bodies. Central to this polymorphic capacity is the conscious self-enactment capacity and I located the history of its evolution from the primate into the communal singing-dancing activity. Here our musically untalented primate ancestors have gain access to the mammalian self-enactment control room providing them with polymorphic bodies. I think Huron’s finding will provide me with some of the ingredient to build this model.
    This is where I see the nature of what is to be human: to be a polymorphic body, to be all kind of animals in the pleotora of worlds it can then crafted with all these bodies.

  • G. Caniaux added an answer in Parametric Methods:
    What is the cross-correlation coefficient threshold in order to be considered statistically significant a non-parametric cross-correlation analysis?

    We are studying the cross correlation between the variations of artificial water reservoirs impounding level with local microseismicity by using Kendall non parametric method. So we like to know when the non-parametric cross correlation coefficient can be considered statistically significant.

    G. Caniaux · Centre National de Recherches Météorologiques

    Dear Fabio,

    As far as I could understand your problem... I would say that statistics is one thing. The physical interpretation of the process at play is another one. It also states the problem of things to compare with statistic (you can always compare quantities even if there is no obvious link between them...). Here i am not able to juge the pertinance of the relationoship between the water level of the dam and the level of sismicity. What I can say is that the statistical test (if of course the data is of good quality) seems to be appropriate (non parametric test; choice of the Kendal correlation);

    Best

  • Vivien Loo asked a question in IP:
    Alternative to LightField for Pylon Princeton CCD camera?

    I've been using LightField for a while now but this program is a failure, from the IP adress attribution to the camera to handling the shutter, or even changing the region of interest...

    I'm looking for an alternative software to control the spectrometer (SP-2-750i) and the CCD camera (PyLoN 400BR eXcelon).

  • Ljubomir Jacić added an answer in Concept Design:
    Applications of the concept "Anti-fragility" ?

    Something is called "anti-fragile" if it benefits from some shocks/attacks/pressure etc upto a certain point and these shocks could make it stronger, for example humans build muscle if work out and have to lift weights for a period of time so their muscles are anti-fragile against physical pressure and work-out. 

    Some things are merely robust or resilient, these are only capable of being strong against shocks upto a point without getting stronger like a glass or a washing-machine. 

    This concept is introduced by Nicholas. N Taleb in a book, I wonder how  we could  make/design/create "anti-fragile "things and apply this concept in various fields.

    Thanks in advance. 

    Ljubomir Jacić · Technical College Požarevac

    Dear @Hossein, one of the application is in system engineering (adaptation and learning)! " Antifragile systems however are defined as systems that adapt after a fault occurs and are not only resilient but also learn from faults and incidents to improve their delivered service level. This paper investigates antifragility in the context of systems engineering and proposes a normative criterion that helps to understand the pre-conditions reaching antifragility."

  • Is there any PCR protocol to increase the chance of amplification of the DNA extracted from acidic water?

    I am working on environmental DNA (eDNA) in aquatic environments. As mentioned by Barnes et al. (2014), environmental conditions influence eDNA stability in aquatic systems.

    Recently, the eDNA that I collected from a small and relatively acidic pond has relatively low yield when extracted, hence it effects the downstream procedures, such as PCR. I am troubleshooting with the PCR step rather than the phenol/chloroform extraction step.

    Is there any PCR protocol that would essentially work on this particular issue (acidity causing degradation of eDNA), ie adding some particular reagents, and or changing temperature settings? Thus far, I could not find any particular solution in the literature.

    Bilgenur Baloglu · National University of Singapore

    Also for BSA, I used to add it for my previous PCR reactions, but not on the environmental DNA yet. It did not make much of a difference for my previous PCR settings, I was usually getting similar bands with/without BSA. 

  • What is the importance of the host organism with which the secondary antibody is synthesized?

    I'm searching for a secondary antibody which I'll use with a primary Rac1 synthesized in rabbit. But what is the importance of the host organism since the secondary antibody will be synthesized by introducing the same primary antibody?

    Can "the ability of the organism to produce a wider range of antibodies to that spesific antigen" be of use to answer this?

    I mean, if chickens were the master organisms in doing recombination during the production of antibodies in B cells (if they were better at producing a more specific antibody), would they be the primary choice?

    Efraim Culfa · Istanbul University

    thank you very much for the valuable information. :)

  • Peter Duesberg asked a question in Paper:
    Renping Zhou is the co-author on this paper with me

    The name of the coauthor is Renping Zhou

  • Ulrike Jung added an answer in RNA:
    Is there any possibility for us to use RNA in fungus in-vitro experiments?

    Hello everyone,

    If dsRNA or siRNA is used in the solution by the in-vitro experiments, what should we do to save or protect the RNAs?

    What should we do when using in-vitro solutions which contain RNA?

    Ulrike Jung · City of Hope National Medical Center

    What do you mean with in vitro experiments? Test tube solution only? Transfected into the fungus cells?

    I would not add ribonuclease inhibitors if you transfect into cells, in the later case I would definitely look into chemical modifications of your RNAs. There is a huge literature spectrum out there if that is what you want to look into.

    In any case make sure you use barrier tips to prevent contamination from your pipettes and make sure never to move with hands/wrists over open tubes/bottles, don't sneeze or cough while something is open and don't talk with open tubes. The main contamination is through aerosol RNAse and in my experience you don't need RNase Zap or masks or anything if your air (avoid open windows, air condition vents etc) "space" is clean.

  • Fairouz Bettayeb added an answer in Rationalization:
    Do you agree that assessment of scientific productivity should be based on the amount of discoveries, inventions and rationalizations?

    Number of publications, citation indexes and other indicators of publication activity should be of secondary importance.

    Examples:

    A new element in the periodic table - a scientific discovery

    The new model of known object - a scientific invention.
    An improved method of solving of known problem - a scientific rationalization.

    Fairouz Bettayeb · Centre de Recherche Scientifique et Technique en Soudage et Contrôle

    It is not easy for every researcher to design and experiment a prototype of his idea or modeling into an accepted scientific invention or patent; the reasons are connected with the available lab. tools, technologies, competencies, organizational and finantial  frameworks, politics.... As mentioned by @Andras the idea is the core of the invention or discovery. So how to measure it and how to protect it from cheating???

  • Emre Can Tüysüz asked a question in Trizol:
    My RNA pellet did not dissolved in RNase free water. What should I do?

    I used Trizol Reagent to isolate RNA. I did what protocol says. FUrthermore, I used 10 ug Glycogen during RNA precipitation step because of small number of cell. After ethanol washing, I dissolved RNA pellet in RNase free water however it did not dissolved. I put my samples into Heat blocker at 58 C. What sould I do?

  • How to find the upstream transcription factors regulating a specific gene?

    I know that a mutant of a gene regulates some biological process. Next I want to find some upstream transcrition factors can regulate this gene. What should I do?

    Daneida Lizarraga · University of California, Berkeley

    There are software that can help you to identify the upstream TFs that may regulate your gene of interest, for example Metacore. This software uses predicted and validated data available in literature. For more details see part 2.9 "Transcription Factor Analyses by MetaCore and PutMiR" of my paper:

    http://www.ncbi.nlm.nih.gov/pubmed/22316170

    Once you identify the putative TF, you should test them, as it was suggested above by Hansi. I think Transfac is also a good option.

    I hope this information helps, let me know if you have questions.

  • Devin Wahl added an answer in HEK293 Cells:
    Why don’t I see a band of my His-tagged protein in western blots?

    I have constructed a pcDNA3 vector with a gene of interest. The DNA sequence of my construct is confirmed and His tag is in frame with my protein. However, when I transfect HEK293 cells with my construct, I can't get a His tag signal in my sample using several different anti-His antibodies. However, the positive control works. Unfortunately the antibody against my protein is not specific so the only way for me to make sure that the protein is expressed is via western blotting or immunofluorescence. My protein is about 60kDa. I don’t know if the problem is with the transfection?
    Any suggestions would be appreciated. Thanks for your help!

    Devin Wahl · National Institute on Aging

    Excellent question, have you checked this website?

    http://www.cellsignal.com/products/primary-antibodies/2365

  • Have you noticed that qualitative medical evolution strictly relates to times when human dissection is performed?

    Have you noticed that, in Human cultures, qualitative leaps strictly relate to times when human dissection is performed?

    I think, of course, of the Egyptian and ancient Greek culture, or the Renaissance times, in which Art in general, and Medical knowledge in particular, evolved in parallel with the improvements of anatomical techniques and cadaveric preservation.

    Can anyone help me find more absolute facts and arguments to proceed with our quest to maintain the habit of human dissection in Modern medical curricula?

    Thank you for your kind collaboration, even if you disagree... 

    Maria Bettencourt Pires · New University of Lisbon

    Thank you for two more, lovely contributions to the issue.

    I was waiting for some free time during the weekend, to answer correctly to colleague  van Zwieten, in strict accordance to his knowledgeable writtings.

    The hands have indeed a lot to do with the intertwining relations between Arts, Humanities and Sciences., because the hands and the evolution and the motion of hands in general are so closely related to the human brain. (Aristote called the hands, the "instrument of the instruments"...) . In account of this, an infinity of ideas coulld arise on this issue . (I will endlessly reffer to Frank Wilson's «The Hand»)

    The idea of the  war injuries is, of course, strictly interrelated  with the necessity of more profound anatomical studies, to perfect and enhance the future of medical practice. I would avoid the issue of commenting on how much Neurology and Neurosurgery evolved in the 20th C, with Mengele's studies during WWII...

    In my Country, traumatology and physiatric wards are mostly devoted to a diversity of injuries from a different war, as we face one of the highest rates of traffic car accidents in Europe. And our "hero" warriors of the trafic have a lot to offer in terms of extravagant injuries, that may well help to improve the general performance of surgeons.

    Thank you Alan Hawk, for your interesting comments.

    I read "The Immortal Life of Hernrietta Lacks", nonstop, during the Summer of 2010 ('), shortly after the first publication, and have been waiting for some free times to review the subject and the immensity of ethical and legal issues, brought by the subject of how a nearly anonymous donnor contributed to some of the greatest scientific  advances in the 20th century, and how the HeLa cells will still be useful in the future.

    Even if this case-study relates to the "perduration" (can't find a less-latin word) of living human material, the question of ethics and of legal demands, in terms of human donations, are of course, one of the main issues in the discussion related to dissection and anonymous contributions to the advances in merdical studies and clinical practice.

  • Sensibility and specificity : who has set the standard scores ?

    Hello, does anybody can tell me the original publication about the norms (criterias) estabilishing sensitivity and specificity scores degree  in a prognostic test ?

    Jean Damasse · L'Institut national d'excellence en santé et en services sociaux

    Hello Mister Eusebi,

    Here is the publication I am refrring to :

    http://www.scireproject.com/sites/default/files/SCIRE4-OM-Full-Ch.pdf

    JD