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  • Gernot Langer asked a question in Biochemical Assays:
    Anyone experienced in biochemical assays of PLA2 activity - without liposomes ?

    I´m about to set up an off-target assay for a PLA2 enzyme and I´m wondering if i do always have to dispense my substrates in liposomes when doing a QRFRET assay

  • Andrei Nikonov added an answer in siRNA:
    Does the siRNA decrease the gene expression?

    Does the siRNA decrease the gene expression?

    Andrei Nikonov · University of Tartu

    @Prekovic: Kevin Morris was not on the list in the paper you posted first :-)

  • Simon Ryu added an answer in Purkinje Cells:
    How to induce in cerebellar slices LTD on purkinje cell using one electrode?

    Hi I would like to induce LTD in PF-PC synapses, but on my set-up I have only one stimulation electrode. 

    Simon Ryu · Seoul National University

    Hi,

    You can use "Conjuctive stimulation(CJ-stim)" to induce cerebellar LTD.

    In CJ-stim, climbing fiber input is replaced with direct depolarization(voltage step) of purkinje neuron.

    This reference shows the protocol of CJ-stim.  ( http://www.ncbi.nlm.nih.gov/pubmed/16234806 , "Cbln1 is essential for synaptic integrity and plasticity in the cerebellum, Nat. Neurosci., 2005")

    You should remember that direct depol. in CJ-stim  just "mimic" physiological depolarization of climbing fiber input, so sometimes the result of LTD induction using CJ-stim could be different from the result of CF-stimulating LTD induction.

    Good luck!

  • Hoda Mcclymont added an answer in Social Media:
    How to build critical mass on social media platforms?

    Does anyone know of any studies or experiences of how to build critical mass on social media platforms? What methods and theories did they use to help them tackle this task?

    Hoda Mcclymont · University of Southern Queensland 

    HI all

    This discussion is wonderful. Thank you so much. I have some new ideas to go on.

  • Minati Singh added an answer in RNA-Seq:
    Does primary human hippocampal culture contain all the neuron types in hippocampus in the brain?

    I would like to distinguish neuron subtypes using single-cell RNA-seq on primary hippocampal neuron culture but not sure how homogenous such cultures are. Any ideas would be appreciated!   

    Minati Singh · University of Iowa

    How about FAC sorting of neuronal  markers and then RNA seq analysis.

  • Which Algorithmic Concept is suitable for building a Self Learning Intelligence in Machine..?

    I  am  using  a Genetic  Programming Concept  in  my  Embedded project... so far  ..  It  works  fine..  But I am still confused  with  some  stages...because  a  lot of  logistics  operations  are  there  in my  project....

    Volker Lohweg · Hochschule Ostwestfalen-Lippe

    A good standard approach is based on Bayesian Decision Trees which are easy to handle an additionally give you the opportunity for an self-learning approach. You will find this topic in many standard textbooks on Machine Learning (e.g. cf. Alpadin: http://mitpress.mit.edu/books/introduction-machine-learning). It is at least a good starting-point.

  • Mahfuz Judeh added an answer in Education:
    What is your view on Take-Home Exams conducted at some Universities?

    There are many ways of testing the knowledge of the students and awarding degrees. While most of the Universities follow a standard Question Pattern (Part-A, Part-B etc) with closed-books and closed-notes, some Universities allow different types of exams like "Open-Book, Open-Notes" exam and "Take-Home" exam etc. In the Open-Book exam, students need not bother to memorize any formula. There will be many questions in the paper, and within the specified time, they should write answers by freely consulting books / notes as they wish. Another interesting option is the "Take-Home" Exam (Final or Mid-term), where the instructors just hand the students a question paper, and they are free to take it to library or home, carefully study the problems, and answer them slowly. Problems will be usually challenging, which require a lot of thinking, but students can think them over and answer slowly. Students are supposed to be ethical in answering take-home exams, and not to consult friends or senior students. I have given an outline! What is your view on these exams? Do they improve the "Teaching-Learning" process? Thanks!

    Mahfuz Judeh · Applied Science Private University

    I consider take home exam is a way of obtaining knowledge; it is a better way rather than recall or memorization.
    There are many benefits of take-home exam, but they are disadvantages as well, especially the risk of inviting improper collaboration or help from others.

  • Souhad El Akoum added an answer in Cell Isolation:
    Cell isolation and culturing them?

    I have some experience in cell isolation and culturing them. However those are mostly short assays (1-2h) and no real stuff. I want to know what culture medium is best for macrofages, lymphocytes, hepatocytes, cardiomyocytes, glial cells...so normal cells no cancer cells! Also can someone suggest methods for isolation of  hepatocytes, cardiomyocytes, glial cells...What enzyme to use for tissue digestion? Some people use colagenase type I while other use IV?

    Next thing is what to do with FCS - some people do heat inactivation while others dont...so what to do? 

    And any other tips that can be useful! I have read may papers and talked to a lot of people that have expirience in cell cultivation but I'm still puzzled...

    Souhad El Akoum · Université de Montréal

    Hi Nikola,

    HEPES is important to keep the pH stable it is very important for cardiomyocytes.

    After digestion centrifugation on percol is recommended but not essential (depending on the age and the rodent strain; I used to work with rat heart). In all cases you have to plate cardiomyocytes to gelatin-coated culture dishes with cardiomyocyte culture media, cited before, supplemented with 5% FBS the first 24h then you can remove FBS. It is recommended to culture it at 0.6 million cell/1 well of a 6 well plate (3ml culture medium) or 0.25 million/well of a 12 well plate (1.5ml culture medium).

  • Arun Vivek added an answer in Reducing Agents:
    Can anyone help with chemical reduction of Ru(III) to Ru(II)?

    Hi, 

    I have a Ru(III) complex with Ered = -0.36 V, and I would like to isolate the Ru(II) complex by reducing Ru(III) chemically, does any one know a suitable reducing agent?

    Thanks in advance.

    Arun Vivek · Loyola College

    If you are working with polypyridine ligands the following answer would help you otherwise pls ignore.

    Inmost of the Ru(II) polypyridine complex formation RuCl3 is used as the metal salt by just using ethylene glycol as solvent and carrying out reflux the reduction can be effected. In the case of ligands like terpyrdine the Ru(III) to Ru(II), can be carried out by using few drops of N-methyl morpholine as the mild reducing agent during complexation in low boiling solvents.  

  • Gareth Abbey added an answer in Cognitive Modeling:
    Are there any new cognitive models of PTSD?

    I'm looking for explanatory cognitive models of PTSD and dissociation. I'm doing research on the relationship between dissociation and PTSD amongst undergraduate students.

    Thank you for your help.

    Gareth Abbey · Kent and Medway NHS and Social Care Partnership Trust

    Hi Justin,

    I am currently finishing a critique of these theories in a paper. Though dissociation is an important issue here, my personal view is that it is merely a symptom of another underlying problem. I say that these theories do not adequately represent dissociation as a construct, but that is because none of the theories adequately represent individual differences in presentation and course of adjustment.

    This is for two reasons: Beliefs and Behaviour.

    Core beliefs are part of schemas (unconsious programs that are triggered by stressful events that determine beliefs, assumptions, and subsequent behaviours). PTSD theories (especially Foa & Rothbaum, 1998) only address beliefs/schemas relating to the danger of the world, and the incompetence of the self - similarly, they only seem to address avoidance behavior consonant with those beliefs (safety behaviours and outright avoidance).

    This is quite an oversight. Jeffery Youngs work identifies many more sets of schemas that have profound implications for adjustment to stressful events. One in particular, the emotion schema, relates to beliefs about the consequences of experiencing and expressing emotion - both on the self.... AND in a social context.

    Emotional processing theory (and by extension, prolonged exposure therapy) is only effective if a person's emotional reactions are sufficently engaged to integrate new information into the fear structure. However, this cannot occur under two conditions:

    1) The person is over engaged. This may lead to panic and then the focus becomes on the fear of fear rather than integrating new information.

    2) The person is under-engaged. This means that the fear structure is not triggered or accessed and no information can be integrated.

    This is important, because emotion schemas may be implicated in how patient's engage with distress, and therapy. It is interesting to note that though trauma-focused treatments are generally very effective, more detailed analyses show that only a moderate number in a group actually recover. Issues regarding therapeutic engagement appear to be related to either over or under engagement in the therapy.

    Though this is not an exhaustive account by any means, what I am trying to suggest is that underyling diatheses related to fear of experience in general might be more useful that the means by which experience is avoided.

  • Minati Singh added an answer in ImageJ:
    I need to know the necessary steps to calibrate my image before measuring neurite outgrowth using ImajeJ?

    I am currently working with primary neuronal cell culture from mouse cerebellum and I'm using NeuroJ -an imageJ plugin- for analysis of neurite outgrowth.

    Minati Singh · University of Iowa

    I was wondering if any anyone has experience in cellular phenotyping in primary  neurons from different brain regions using RNA seq method

  • Eric Baird asked a question in Wormholes:
    Does inflating a Planck-scale wormhole to macroscopic size add internal space and "transit capacity", or do its internal measurements remain tiny?

    What does it actually mean when we talk about “inflating” a wormhole? If we find a Planck-scale natural wormhole, and we cram exotic matter into its two mouths to stretch it up to, say, one metre wide, then the wormhole may nominally now be a metre across … but have we actually added any additional useful space to the throat interior, or have we taken a throat that only has a fixed amount of internal space and "stretched" that fixed space, so that although it's now nominally one metre across, the internal measurement (and the wormhole's “capacity” as measured with internal rulers) might still be Planck-scale?

    Would inflation be adding more space and more useful “transit capacity” to the wormhole throat, or would we still have the original Planck-scale throat, inhabiting a distorted and stretched region of space in which everything is rescaled and magnified?

  • Antonio Ariza added an answer in Column:
    Problem in purifying protein through Ni Column

    I am trying to purify my protein through Ni column but my hydrophobic protein is not binding to my Ni Column.. Anyone can help me? Thanks

    Antonio Ariza · University of Oxford

    I guess that if your protein is hydrophobic and you're not using any detergents it might not even be soluble in the aqueous solutions used for Ni-NTA in the first place. Have you verified your problem is in solution in your supernatant after cell lysis? It might actually be in the pellet as an insoluble fraction or even inclusion bodies, in which case you obviously wouldn't see it binding to your Ni-column. If it is really hydrophobic you should indeed use some detergent and you could also check how people purify membrane proteins, as the buffers they use might be a good starting point for you.

  • Odenu Iyede asked a question in EViews:
    Using EViews to establish the differential in sensitivity between variables A and B to variable C

    I am currently working on a macroeconometric model and one of the goals is to determine if response of variable A  to variable C differs from response of Variable B  to variable C. How can I address the issue with the use of EViews.

  • What would you do if you received a report from the reviewer of your paper asking for a major revision, with which you completely disagree?
    What would be your response? Would you appeal to the editor by asking him/her to make the final decision, would you be weak and amend what you believe is right, would you argue back with facts, would you re-submit the paper to another journal?
    Mahfuz Judeh · Applied Science Private University

    Dear Jean,
    I really feel sorry for what happened to you in your research; it is a frustrating situation. This rarely can happen to any researcher. Anyhow, I think it could have been better for you if you had not known about the reviewer.
    Reviewers must disclose to editors any conflicts of interest that could bias their opinions, either with or against, on the manuscript.

  • Richa Jaiswal added an answer in Laminin:
    Do MCF7 cells need special coating on the glass coverslip?

    My cells don't spread well on acid washed coverslips, I have no clue why? Do I need to coat them with laminin or fibronectin or anything else? 

    Richa Jaiswal · Brandeis University

    No Ian Cartwright, I am seeding cells for immunofluorescence microscopy and they look more rounded even after 24 hours of seeding. That's why I was wondering if they need some special coating for them to spread well. Thanks for your response though.

  • Can someone help me with appliance scheduling in smart grid?

    I have a list of appliances to be scheduled for a day. The appliances should be scheduled during low peak price period inorder to avoid peak demands. I am using optimization solvers from matlab like linear programming solvers and binary integer programming. I dont seem to get any optimized results. I find that the appliances are getting switched during peak price period. Can someone help me in solving this or suggest any other solvers?

    Malar Vizhi Kanniappan · Sri Sivasubramaniya Nadar College of Engineering

    Dear Ayodeji samson Ogunjuyigbe,

    Thank Your sir. 

  • Antonio Ariza added an answer in SUMO:
    Is anyone familiar with SUMO fusion Protein Cleavage?

    I expressed an alpha-fucosidase in a pET SUMO vector that SUMO fusion protein is cleaved with a specific protease after expression.
    The problem is that protease was not efficient to remove SUMO. I have tried all possibilities and checked all buffers and salt concentrations but SUMO wasn't cleaved. Does anyone have any suggestions how I can cleave this fusion protein? Thanks!

    Antonio Ariza · University of Oxford

    Although SUMO protease is salt sensitive, it is a lot less sensitive than TEV protease for instance and can even be used at 1M NaCl as long as you provide a huge excess of protease. Since my Ni-NTA buffers tend to have at least 500 mM NaCl in them, I usually cleave ny protein samples at the same time as I dialyse out the imidazole and reduce the NaCl concentration. I put my Ni-NTA elution sample into a dialysis bag and dialyse it for one hour into buffer containing 20 mM TRIS pH 8.0 + 150 mM NaCl + 1 mM DTT. This will radically reduce the NaCl and imidazole concentration inside the dialysis bag, which I then remove from the dialysis beaker/bucket. I add the appropriate amount of SUMO protease into the dialysis bag and put it into a new container with fresh buffer, which I leave at 4oC overnigth with gentle stirring. Some of my proteins used to precipitate a little bit during this step, but that was not a problem with the tag removal but because they don't like buffers with low ionic strength. To overcome this I kept the NaCl concentration between 300 and 500 mM for these proteins and added an excess of SUMO protease. BTW, if you make your own protease (buying them is very expensive) make sure you keep them in small aliquots at -80oC, thawing and refreezing larger aliquots several times can kill their activity.

  • Jay Kant Yadav asked a question in Neurotoxicity:
    Can any one suggest an appropriate neuronal cell line for studying the amyloid mediated neurotoxicity

    We are working with several amyloidogenic peptides and world like to investigate their neurotoxic effect.  

  • I expect the opinion from the RG Professors regarding the unethical practices in the conference presentations.

    Dear Sir,

    Ten years back in conference presentations the presenter used to hide the identity of the diseased persons if they were showing the picture of the diseased person by covering a black patch around his eyes. This was also the practice in the medical books. In recent conferences, pictures of the diseased persons are presented without hiding the identification. Is it fair on the part of the presenter to do so?
    Dr. Shanmugasundaram P

    Palani Shanmugasundaram · Vels University

    Dear Professor András Bozsik •

    I think Anatomical acts of every country deals about it. But I consulted some lawyers in India they have no answer to this question. I thank all the professors about the deep concern they showed to my question.
    Dr. Shanmugasundaram P.

  • Do you work with a protein that is SDS-resistant?
    My lab is working on constructing a database of SDS-resistant (i.e. kinetically stable) proteins. By "SDS-resistant", I am referring to those proteins that will not be denatured by SDS without heating. If you would like us to include your SDS-resistant protein, please provide the name, Uniprot #, and reference (if available) that validate its SDS-resistance. Our goal is to make the database accessible online to the scientific community.
    Wilfredo Colón · Rensselaer Polytechnic Institute

    Hi Ron and Dina:

    Thank you for your responses. This is a great forum to learn more about this issue. SDS-PAGE is such a common technique and it is so easy to test SDS-resistance, I hope to keep learning and expanding our database of kinetically stable proteins. The biological and pathological implications of this property are fascinating. Best, Freddy

  • Kuldeep Dhama asked a question in Research Papers:
    In drafting and publishing a research paper, who among all authors could be most advanatgeous

    Among all authors of a research paper which author can be most reliable for acceptance of a paper at the earliest feasible time 

  • Alain Celzard added an answer in Activated Carbon:
    What is the best method and material for preparation of activated carbon?

    For pyrolysis of waste plastic.

    Alain Celzard · University of Lorraine

    Given that most plastics have a carbon yield near zero when pyrolysed, as most of the material decomposes as gases, chemical activation, especially with H3PO4, will give the highest yield. Moreover, it is an easy and cheap technique, which can be carried out at moderate temperature (around 500°C) and even directly in air. See papers in my RG profile for details.

    Alain

  • Simone Farinelli added an answer in Bhutan:
    Please confirm me ID of the following Lemyra sp. (Erebidae; Arctiinae).

    Collected from Bhutan. No. 1 is a probably a female and 2a & 2b ia a male sp.

    Simone Farinelli · Centralschweizerische Kraftwerke AG, Luzern, Switzerland

    No

  • Alain Celzard added an answer in Activated Carbon:
    What is the best way to modified an activated carbon?

    What is the best way to modified activated carbon? I want to have high surface area and to support the metal.

    Alain Celzard · University of Lorraine

    If you already have an activated carbon, and want to modify it for increasing its surface area, you have to pay attention to 3 things:

    1. which kind of porosity do you want ? If your material is already highly microporous, a further activation may create more mesopores and make its surface area decrease. This may happen with physical activation, e.g. with steam.

    2. do you mind having impurities inside ? Chemical activation, e.g. with KOH, may create much more micropores, but even after extensive washing, traces of K may remain.

    3. is the pH important ? KOH and H3PO4 activation will lead to basic and acid carbons, respectively. Therefore, their point of zero charge will be different and, depending on the pH at which you'll do the metal impregnation, the ions may be preferentially adsorbed or not by the carbon surface.
    Alain

  • Hossein Haddad added an answer in Piles:
    Can anyone help with references on the topic of pile setup, or long-term gain in pile capacity?

    I am interested to study the long-term increase in capacity for different categories of piles in different types of geomaterials. Right now, I am trying to collect and review all the relevant literature on this topic in order to find if there are any gaps that could be plugged, or some investigations could be conducted towards improved understanding. I found following summary information on this topic. A help in finding the related literature will be appreciated.

    Pile resistance in long-term behavior involves setup and increased capacity over time. Effective stress (or the drained characteristics) governs the long-term behavior of piles, especially in clayey soils, following dissipation of excess porewater  pressures. In addition, ageing and other rheological effects (i.e., pile "freeze", thixotropy) can occur in clays, silts, and sands following the pile installation. Pile setup has two major components: (1) short-term setup caused by the dissipation over time of the excess pore water pressures generated during the pile installation, (2) and long-term setup, i.e., additional gain in strength over time at constant effective stress due to other factors, such as secondary compression (creep), bonding, and/or ageing (Konrad and Roy, 1987; Whittle and Sutabutr, 1999; Svinkin and Skov, 2000; Randolph, 2003; Karlsrud and Haugen, 1985; Schmertmann, 1991; Basu et al., 2013). Basu et al. (2013) indicated that the mechanisms of setup and the setup periods are different for fine-grained and coarse-grained soils, because of the drainage characteristics and other intrinsic properties of these geomaterials that affect the soil-structure interactions and their response to the loading.

    Hossein Haddad · Amirkabir University of Technology

    You can find useful information in Wagner and Komurka report about pile setup.

    minds.wisconsin.edu/bitstream/handle/1793/6881/03%2005.pdf

  • Ankita Gupta added an answer in PEG Precipitation:
    M13 ssDNA preparation

    We obtained M13 ssDNA from NEB and transformed suitable host bacterial strain followed by phage preparation from the same. Phage was then used for purification of ssDNA by PEG precipitation and phenol extraction (detailed protocol was followed as given in Molecular Cloning by Sambrook and Russell) and was used as a substrate for enzyme assay. There was no activity found. However, when M13 ssDNA supplied by NEB was used for the assay, enzyme activity was detected. When both DNA samples (isolated in lab and supplied by NEB) were run on the gel, one isolated in lab was found to run slower than that from NEB (image attached). Is there something wrong with the way I am isolating ssDNA even though M13 phage used for this was prepared from ssDNA supplied by NEB?

    Ankita Gupta · Indian Institute of Technology Bombay

    Thank you very much!