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  • Tommy Lu asked a question in Confocal Microscopy:
    How to minimise bleaching on confocal microscopy?

    I've used the NIKON A1 inverted confocal microscopy a couple of times and I noticed the red stain at 540/565 nm gets bleached within seconds. The objective I use is an oil 60x. Could anybody please let me know how this can be fixed? Massive thanks!

  • Morten Suppli added an answer in Prostate Cancer:
    Any thoughts or feedback on the manuscript from the Advanced Prostate Cancer Consensus Conference (APCCC, March 2015, St. Gallen)?

    In March 2015 the first Advanced Prostate Cancer Consensus Conference (APCCC) has taken place in St.Gallen, Switzerland. A panel of international prostate cancer experts have discussed and voted on pre-defined consensus questions relating to management of men with advanced prostate cancer. The manuscript was recently published (Annals of Oncology, open access) and we would be very interested to hear thoughts or get feedback on this.

    Morten Suppli · Rigshospitalet

    Regarding the discussion of continuous or intermittent ADT you miss an important reference. Hussein et al. NEJM 2013. The Niraula review cited in this paper has only included an abstract of the SWOG trial. When discussing iADT we should consider that the actual overall survival in both groups is longer than expected and how this affect the non-inferiority margin. In the Hussein paper non-inferiority margin was an HR of 1.2. Is this difference still acceptable?


  • Soosai Manickam added an answer in Hep G2 Cells:
    Can anyone recommend a good quick spheroid formation protocol in 6- well plates?

    Hi, I am trying to generate a spheroid to culture HepG2 cells , and my objective is to evaluate the effect of different culture conditions on the marker expression. Not really on the optimisation part here then. My plan is to plate them in 6-well plates so I'll have enough cells (my downstream analysis is western blot).

    I knew there's a commercially available plates out there like Corning/Nunc sphere plate but they are quite pricey.

    Does anyone here make their own low attachment plates (using 6 well plates) to make spheroids and was it good?

    Soosai Manickam · Christian Medical College Vellore

    Try non-treated culture plates (suspension culture plates) or 2% agar coated plate and increase seeding density.

    God Bless...

  • Shantamoy Guha added an answer in Landslides:
    Can stream gradient indices apply to identify landslide prone areas?

    Any researcher who have knowledge about this can answer.

    Shantamoy Guha · Indian Institute of Technology Gandhinagar

    Steep hillslope is mostly susceptible for the landslide processes, but it also is affected by geology (rocktype and structure). Identification of the streams in hillslope with the help of DEM is also a difficult task. With the help of topographical maps one can easily identify the individual streams and their gradient.

    You can try to fit some analytical models with the help of existing landslide events data and stream gradient. These models will be different in different geological area. So simply with the help of stream gradient you can not identify the landslide prone areas. I have attached an article. You can try to use this.

  • n Arunachelam added an answer in Geopolymer Concrete:
    Why is the density of the ambient cured geoplymer concrete higher than hot air cured concrete?

    reason why this variation happens in geopolymer concrete

    n Arunachelam · Gandhigram Rural Institute

    Source material is Class F Fly ash and sodium based activator solution

  • Paul M.W. Hackett added an answer in Metaphysics:
    What is perceptual mereology?
    Is anyone aware of literature that considers what could perhaps be thought of as perceptual mereology? What I mean by this is a parts-whole account of how we perceive and understand our worlds, how we bring the parts we apprehend together to form the whole worlds as meaningful experiences? This question arose out of another question I posted and I wanted to share this with a wider audience.
    Paul M.W. Hackett · Emerson College

    Thanks Michael.

  • Jesús S. Aguilar-Ruiz added an answer in Genetic Algorithm:
    Anyone familiar with Real Crossover techniques in genetic algorithms?

    Can anyone tell me from where I can download the research papers related to the real crossover techniques used in the genetic programming.I need the very first ones in order to understand the basics of real crossover

    Thank you

  • Taha Mattar added an answer in Steel:
    Dear members, Does anyone know what is the grade of steel, based on the following elements composition?

    Does anyone know what is the grade of steel, based on the following elements composition?

    Element       Percentage (wt) %

    C                    <0.5

    S                    <0.5

    Si                     3.47

    Mn                  0.75

    Ni                   <0.1

    P                    <0.1

    Cr                    0.89

    Cu                   0.12

    Fe                   90.71

    Mo                 0.2

    B                    0.63

    Al                   0.21

    Ti                   0.21

    V                    <0.1

    W                  <0.1

    Pb                 <0.1

    Nb                <0.1

    Taha Mattar · Central Metallurgical Research and Development Institute

    Free cutting steel

  • Daniel Yero added an answer in Klebsiella pneumoniae:
    I dont understand the difference between a clonal complex and clonal group in Klebsiella pneumoniae clones. Somebody can help me?

    for example:

    St101 in some artciles speak about it as a clonla group(CC) 101, other directly is a clonal complex (CG) 101.

    ST14 in some artcles its finding in CC15 and CG15, but in other it seams is CC14 and CG15.

    I have to others STs: 450, and 1233 that have 2 and 1 Single locus variant (SLV)) but wiothout any information about it CC and CG.

    Daniel Yero · Autonomous University of Barcelona

    Indeed, you are right! In bacterial evolution there are so many errors or misconceptions. My experience is with N. meningitidis MLST (the first MLST scheme) By definition in a clonal group the isolates shared common ancestry. Then, a clonal complex is a clonal group as just as the ST is a clonal group. In your paper a group of clonal complex are named as a clonal group because they probably have a common ancestor and the CG takes de ST number of the founder CC by the eBURST analysis. The concept is in the paper (new for me) "We further defined clonal groups (CGs) of CC37, as this CC tended to merge, constituting a group of unrelated STs rather than one diversified from a single common ancestor".
    if I understood well, for these authors since a CC is more clonal (all ST emerged from a central ST as SLV or DLV) and the CC37 is more diverse (but still a clonal complex), they defined CGs within the CC37. In my opinion the CC37 is the result of smaller CCs (called here CGs) connected through SLVs strains (probable due to recombination events).

  • Alok Pandey asked a question in Forecasting:
    Kindly suggest some good papers on K- Nearest Neighbor (K-NN) regression used in the field of forecasting?

    I want to know more about the way this method has been used for forecasting. 

  • Eugene F Kislyakov added an answer in Cognitive Systems:
    Is Chalmers' so-called "hard problem" in consciousness real?

    In his 2014 book "Consciousness and the Brain: Deciphering How the Brain Codes Our Thoughts" Stanislas Dehaene wrote "Chalmers, a philosopher of the University of Arizona, is famous for introducing a distinction between the easy and the hard problems. The easy problem of consciousness, he argues, consists in explaining the many functions of the brain: how do we recognize a face, a word, or a landscape? How do we extract information form the senses and use it to guide our behavior? How do we generate sentences to describe what we feel?

    “Although all these questions are associated with consciousness,” Chalmers argues, “they all concern the objective mechanisms of the cognitive system, and consequently, we have every reason to expect that continued work in cognitive psychology and neuroscience will answer them. By contrast the hard problem is the “question of how physical processes in the brain give rise to subjective experience … the way things feel for the subject. When we see for example, we experience visual sensations, such as that of vivid blue. Or think of the ineffable sound of a distant oboe, the agony of an intense pain, the sparkle of happiness or the meditative quality of a moment lost in thought … It is these phenomena that poses the real mystery of the mind”."

    Stanislas Dehaene's opinion is "that Chalmers swapped the labels: it is the “easy” problem that is hard, while the “hard” problem just seems hard because it engages ill-defined intuitions. Once our intuition is educated by cognitive neuroscience and computer simulations, Chalmers’ “hard problem” will evaporate".

    Personally, I agree with Stanislas Dehaene's opinion.

    Eugene F Kislyakov · Belarusian State University

    As I know, Andrey, last Freud's words were that they all (all their history) are mads.

  • Shreyas Lingadahalli asked a question in LNCaP Cells:
    LNCaP cells clumping up on Matrigel ?

    Hello all,

    I am try to grow LNCaP cells on BD Matrigel to perform some invasion and migration assays. However, my cells gets clumped as u can see in the attached in pic  (they look fine on uncoated plates/wells) . Matrigel was diluted 1:100 and allowed to solidify for 3 hours in 370C  incubator. 

    Any insights, tips or hints will be much appreciated. 

    Thank you 


  • Rex Bringula added an answer in Path Analysis:
    What are the advantages of partial least squares path analysis versus AMOS-LISREL type search algorithms?

    Advantages of the two techniques when conducting SEM

    Rex Bringula · University of the East

    Dear Theo,

       They are legitimate responses. However, for example, when I asked the respondents' Internet and computer skills (1 - not skilled to 5 - highly skilled), the data tended to be positively skewed. So, I decided to exclude Skills as a variable and made it as a profile.

       (As to the button, sure no worries. I didnt know that there was a rate button. :D)

    Thank you,


  • Boris Ivanov Evstatiev added an answer in Transmission Line:
    How can I relate a transmission line's propagation constant with the ABCD parameters?

    Consider a transmission line with Z0=R+jXL and Y0=G+jXC. The propagation constant and the characteristic impedance are:



    Considering the transmission line is symmetric, we regard the two-port as a T-network with Z0/2 on both sides. Then the ABCD parameters are:




    For a reciprocal two-port Z0 and γ are:



    But here comes the problem. If ZC=Sqrt(Z0\Y0) then Z0Y0=0 and this means that γ=0. Something is wrong and i don't understand what.

    Boris Ivanov Evstatiev · University of Ruse Angel Kanchev

    Thank you. That makes sense.

  • Aman Singh added an answer in HMM:
    Which tool is best to use for implementing hidden markov model and how ?

    I want to use HMM for prediction based on state and emission probabilities 

    Aman Singh · Lovely Professional University

    Respected Sir (O. Akay),

    Thanks for the reply.

    As, I am just a beginner. Kindly tell me should I go with per-defined functions in Statistics and Machine Learning Toolbox or with the HMM packages available at the link.   

  • Ray Kruse Iles added an answer in Oxytocin:
    Does anyone have experience of oxytocin urine samples? Can you recommend any labs and know the cost of analysis in England GB? It is needed urgently!

    Hi All

    I am conducting an RCT for my PhD measuring Oxytocin Levels in mothers and their babies. After my previous post I have decided to do this with Urine samples as it feels more ethical than Saliva samples as then baby and mother can still eat. I need to cost the analysis of these samples and I was wondering if anyone else has done this analysis, particularly in England, GB? Does anyone know the cost of having the samples analysed? I am surmising that the kits are not DIY as all the research talks about centrifugal force in the lab? Any advice, recommendations would be gratefully received.

    Ray Kruse Iles · MAP Diagnostics Ltd UK

    Hi Kate - sorry for delay MAP Diagnostics is in Welwyn Garden City North of London

    my work email is ray@mapdiagnostics.co.uk

  • Mohammad Ishaq added an answer in Thapsigargin:
    What ratiometric method can be used to detect cytosolic calcium release after 12 hours of stimulation?

    Hello guys, I am doing some experiments which involve increase of cytosolic calcium. At present i do not know what is the source of this increased calcium in cytosol. So i just wanted to confirm what are the best methods/dyes to study the organelle (mitochondria or ER) specific calcium release.

    Also i am using Fluo-3AM for calcium measurement and i have observed that increase in cytosolic calcium was detected after 6 hours of stimulation which continues to rise up to 24 hours (by flow cytometry). I used Thapsigargin as Positive control.However, I am being told that fluo-3AM signal is not only dependent on calcium concentration but also on cell volume and granularity.I was recommended to do the ratio-metric analysis. So please suggest is it possible to do the ratio-metric analysis at longer time points after stimulation. I hope this much experimental is enough for some description will help you to give some good suggestions. Thank you in advance.      

    Mohammad Ishaq · Institute of Microbial Technology

    Thank you sir for your suggestion. But i want to know which method is better to analyze the calcium efflux into cytosol. Is it better to use fluorimetry based methods or microscpy. Thank you once again

  • Gaurav Sharma added an answer in Pfam:
    How to remove overlapping hits in hmmscan output against any profile database?

    Whenever we do hmmscan against any sequence profile database such as Pfam, TIGR or CAZY, we always get overlapping domain hits either from the same profile or the different profile. How to remove these overlapping hits from the result file? 

    Gaurav Sharma · Institute of Microbial Technology

    Dear Ashmit,

    I hope you are familiar with hmmscan domtblout result. Many times some domains are fully contained within the other [Please see the highlighted area per protein in attached file]. It is hard to parse them completely. I have checked some scripts available online to parse the results but all in vain till now. 

    Thanks in anticipation.

  • Fuad Bahram added an answer in Immunohistochemistry:
    Is there a marker or stain of endothelial cells that will cross the placental barrier?

    The purpose is to image whole mount cleared mouse embryo heads (E13.5-E18.5).  I am concerned that standard methods of immunohistochemistry will not work for whole mount specimens of this size.

    Fuad Bahram

    Try to stain VEGFR2 or/and 3 . We have not been working with placenta but but som both organs and mouse EC. Check this one of my publication you will find some of answers you need. 


  • Ruben Alvarez-Fernandez added an answer in cDNA Synthesis:
    Does cDNA synthesized using a cDNA synthesis kit for Real Time PCR use works in End Point Conventional RT-PCR?

    I have synthesized cDNA from 1ug of RNA using Quantitech Reverse Transcriptase RT Kit from Qiagen which is for Real Time PCR use. But in out lab we have Conventional RT-PCR. Can i use this cDNA for PCR? I tried with this cDNA for Positive control several times, varying different conditions and gradient method but could not get any band. I used Top Taq Master mix from Qiagen

    Ruben Alvarez-Fernandez · University of Essex


    The kit instructions read that it is optimised for qPCR, but there are so many master mixes in the market that very likely they optimised it for their own qPCR mixes. It produces single-stranded cDNA using poly-dT and random hexamers (so the cDNA covers both the 3' and 5' regions of the mRNA, and due to the random hexamers it reverse transcribes other RNA's as well) of at least up to 12.5 Kb. Also, it cleans the mix from genomic DNA.

    In other words, yes, your cDNA should perform fantastic in RT-PCR as well.

    To troubleshoot, check these questions:

    - does the RT-PCR mix work with other templates/primers?

    - do the primers you use amplify other templates? If no, your primers are off or badly designed. If yes, either your cDNA is off or you are using primers that bind to genomic but not to mRNA.

    - if the answer above is yes and the primers are correct and bind to mRNA, what steps did you follow to ensure integrity of your RNA? If it is degraded (in this case you better discard it for qPCR or semi-quantitative RT-PCR) and you are trying primers that span a big amplicon (>1 Kb), very likely you won't get much amplification if at all.

    Good luck,


  • Serge V. Shil’ko added an answer in Physical Metallurgy:
    Are there any issues with performing compression tests on samples with square cross-section?

    The sample (copper) is rolled to about 6 mm thickness. I wanted to do compression testing on samples along the rolling direction. Since the sample thickness was to small I was left in a predicament to use EDM wire cutting to prepare samples with square cross-section (6 mm) and length 9 mm. I could not cut circular sample since the gripping width is too small. I am primarily interested in studying the microstructure of copper with varying compressive strain along length. Hence are there technically any issues in performing compression tests on these samples. 

    Serge V. Shil’ko · National Academy of Sciences of Belarus

    Jointly with point of view of John Butlerthe, in developing  detail (microlevel) analysis of plasticity and failure processes, the book "Micromechanics and Nanosimulation of Metals and Composites: Advanced Methods ... by Siegfried Schmauder and Leon Mishnaevsky as well as papers of Prof. V. Panin with cooworkers on physical mesomechanics (f.e. Panin V.E. etc Foundations of physical mesomechanics of structurally...) may be recommended for your attention. 

  • Sharon Balasubramanian asked a question in Fracture:
    Is it possible to  obtain the result of the postulation below?

    "On-set of fracture is said to occur for any laminate when COD of  a node just behind the crack tip reaches a value that corresponds to failure  of a laminate  made of same material for which COD is known for the same mode of expected failure" Suppose if we know the fracture load and COD of ±450( Eg:1mm),is it possible to predict the fracture load of 30 for the same COD of± 450(Eg:1mm) in 300

  • Tomasz Wejrzanowski added an answer in Metallurgy:
    Can anyone suggest a organic binder instead of conventional inorganic binders for metal powder compaction in cold condition?

    I want to use organic binder instead of inorganic binder for metal powder compaction for comparative study between organic binder to other conventional inorganic binders. 

    Tomasz Wejrzanowski · Warsaw University of Technology

    Did you try CMC or Polyox. We are using it for tape casting of Ni-based green tapes.

  • Juliette Mouries added an answer in Oral Tolerance:
    Do I need to renew water for OVA water feeding ?

    I want to move from OVA gavage to OVA in drinking water in a model of oral tolerance. Should I change the OVA containing water every other day or is it stable enough to stay an entire week ?

    Juliette Mouries · IEO - Istituto Europeo di Oncologia

    Thanks a lot,

    this is exactly the kind of advice I was looking for !

  • Jochen Wilhelm added an answer in T-Test:
    Why do we use t-test when one of its assumptions is violated? Isn’t it better to use Mann-whitney U, when there is no equality in variances?

    Hi there, Considering the fact that “if the Levene's test produces a significant result (i.e., p is less than 0.05) then we use the lower line that is labeled equal variances are not assumed. In this case, the result is based on a correction for the lack of homogeneity of variance.” How is it possible to use t-test when one of its assumptions is violated? Isn’t it better to use Mann whitney U, when there is no equality in variances?

    Jochen Wilhelm · Justus-Liebig-Universität Gießen

    Two things are generelly forgotten or ignored:

    1) Data analysis is always basd on some assumptions, which provide a frame or context which makes it possible at all to interpret values in the first place. The normal distribution is not a natural phenomenon, like a circle or a straight line are not natural phenomenons. These are models, abstractions, logical tools. So it is not and can never be the question whether or not some *real* variable has a normal distribution. It does not - we know this. Any "non-significant" test of the null hypothesis that real data is from a normal distribution is thus a known false-negative. The relevant question is if the difference to this model is so severe that the conclusions based on calulations that are based on such an assumption may be considerably compromised or misleading. Judging this is not always easy, and using anather hypothesis test does by no means solve this problem. To add to Timothy's answer: even a significant result does not tell you anything useful (the detected deviation may not be relevant - so this may be a "practically" false-positive).

    2) Hypothesis tests are a decision-theoretic tool that are based on "long-run properties" and expected consequences of the decisions (that may be correct or wrong). Their value depends on the strategy used to produce a decision. The desired error-rates are held only when this strategy is appplied. This strategy requires that pretty much of the properties of the data are known in advance. One may need preliminary experiments and experience to get a good-enough idea about these things (like distribution, variance, effect sizes). The actual test then requires to use new data, that has not been used before, for instance to select the probability model (the test). If one changes this strategy to: "I will first collect some data, then I will see how it is distributed, according to this I select a transformation (log, srqt,...) or a test (t-test, U-test, ...), finally I will perform the test using this data" then the decision to reject the null hypothesis based on alpha will not even control the type-I error-rate.* So it is completely inapprehensible why all this effort is taken when the only partly useful property of a hypothesis test kicked off.


    *not mentioning the lack of control of the type-II error, what makes tetsing quite useless. Fisher's significance testing does not require and is not based on the any control of error-rates at all. He said that "We shall not often be astray if we draw a conventional line at 0.05 [for rejecting the null]"(1). This is not a formal control of an error-rate, and it is not meant as a rule to always reject the null whenever p<0.05.With "being astray" Fisher did not mean the fact whether or not we correctly identified any "true effect (or difference)" but the fact that we decide to do further research that will likely be unsuccessful - because the noise is too large or the effect size is too small to be handeled efficiently enough in subsequent experiments.

    (1) Fisher, RA: Statistical Methods for Research Workers, 1925, p.80.

  • Jesús S. Aguilar-Ruiz added an answer in Genetic Algorithm:
    Are genetic algortihms worth of it?

    I just came to know from a lecture that the genetic algorithms are not mathematically logical means they do not have sound logical background especially when we turn to the real coded ones which are not even in analog with the biological evolution.What are thoughts on it?

  • Stamatis Zogaris asked a question in Salinity:
    Ever seen a mass mortality event of ranid frogs in a brackish lagoon?

    We documented a mass mortality event involving 2000+ sub-adult (almost fully grown) Ranid frogs at a brackish lagoon on Samothraki Island (Greece) just a few days ago (the Lagoon is called Koufki Lagoon, adjacent to Agios Andreas Lagoon SW of Kamariotissa port). Most dead and many living frogs were concentrated at areas of the lagoon that had slightly lower salinity - and water salinity may be rising (due to rapid drying). We have bio-samples and water samples and measurements. What we are interested in, is if anyone has seen this kind of event before in any kind of fresh or brackish waterbody? Any bibliographic support would be much appreciated.

  • J.W. Geus added an answer in Hydrogenation:
    Where can I find a procedure for a Platinum Oxide catalyzed hydrogenation reaction?

    I am interested in the use of steel bombs and their setup.

    J.W. Geus · Universiteit Utrecht

    Already at low temperatures, e.g., room temperature, platinum oxide, which is thermodynamically not stable is reduced by gaseous hydrogen. Oxygen chemisorbed on metallic platinum is also rapidly removed by contact with gaseous hydrogen. Hydrogenation will be over metallic platinum.

  • Hywel Coleman added an answer in Law and Courts:
    Are there any cases of judgements by law courts which affect curriculum matters (English language or more generally)?

    I am currently writing a paper together with an Indonesian colleague which discusses a case in which the Constitutional Court of Indonesia banned a school programme which had CLIL-like characteristics (CLIL = content and language integrated learning). Is anyone aware of other cases anywhere in the world where LAW COURTS (not legislators in parliaments) made decisions which impacted on education policy and practice, particularly in relation to curriculum? Our interest is not restricted only to the teaching of English. Thank you.

    Hywel Coleman · University of Leeds

    Dear Meinert. Thanks for informing us about this intriguing case. I have spent an afternoon trying to identify the paper by Prof von Hentig which you refer to but without success. (My German is extremely limited.) Therefore when we refer to this case in our paper we will probably give the source as 'Meyer (personal communication)'.