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  • Richard Goodin added an answer in Peptides:
    A very basic question but how do mimotopes work? I know what they are but what information do you get from the binding of a peptide to a protein?

    I have REPLi-K or REPLi-K/R peptides and we are using them to look at binding to a specific protein? I get data but don't know the exact information it is telling me except that it is a mimotope assay.

    Richard Goodin · Patheon

    Louise, For information on protein 3D structure, go to the National Library of medicine and look up your target protein. You can see where your binding site is located and can possibly locate new binding sites that are accessable and not internal or masked.

  • Angel Jurado asked a question in Scientific Publishing:
    Has anyone used encyclopedias to look up for info in the Organic Chemistry / Chemical Engineer Fields?? Do you have comments on that?

    Hi Everyone

    I’m currently working on a summer project to try to understand the best ways to upgrade the user experience of one of the biggest scientific publishers in the world. I’m particularly interested in understanding how scientists interact with publications and encyclopedias in organic chemistry/chemical engineer fields, so I can give them some advice on how to best improve accessibility and usability of the scientific content they put out there. I would truly appreciate your insights on this matter and I’m seeking researchers that currently work in R&D departments of Pharma or Chemical organizations in the US, UK, Germany, France or Japan.  We will pay you for a 1-hour telephone interview, and your identity is kept confidential.  If you would like to participate in this initiative, please contact me through private message or send me and email to angel.jurado@rutgers.edu

  • Akira Kanda added an answer in Philosophy Of Science:
    Is the economic collapse – a black hole syndrome?
    Mathematics justifies that greed and extreme selfishness cause economic collapse of societies.

    Any system that is established from relations from different groups and the forces created thereof, continues to exist if the relations remain fair and the forces that are created from the relations to keep the system alive remain on balance and valid to all parts. When one of the forces dominates to the extent of diminishing the strength or eliminating the powers of others, then only a force of pulling towards the dominant entity remains and that leads to the collapse of the system - this is what I call it the black hole syndrome.

    What do you think, is greed and extreme selfishness the cause of economic system collapse?
    Akira Kanda · University of Toronto

    Today, rich people are young, they are the generation X. Immature godless kids screwing up the entire world. They grew up watching "Law and Order" TV show every day made to create Neo Nazi pseudo intellectuals. Yes they are experts on the difference between debt and deficits.

    In the old time, people did not cross certain lines for the fear of life after death. These kids are the creation of this masonic project of destroying all religions world wide. Masons want to rule the world and religious view is a big hindrance for them. This is why we keep seeing "religious" extremists terrorists created by them. Extreme religious terrorism is the department of Puritanism (Christian Libertarianism).  as the history shows

  • Barbara Sawicka added an answer in Yoga:
    How can yoga affect health? Can it deal with some diseases?

    I collect Data about yoga if some one has informations about it

    Barbara Sawicka · University of Life Sciences in Lublin

    The biggest advantage of yoga is to alleviate the symptoms of stress. With a slow, precise movements, positions requiring concentration and breathing in a controlled manner yoga moves our thoughts from the problems of everyday life. While exercising, we focus on yourself and your body, and thus purify the mind

  • Gedion Firdawek added an answer in Public Hospitals:
    Can u give me a good research that has been conducted on budgeting and budgetary control system in a hospital?

    Can u give me a good research that has been conducted on budgeting and budgetary control system in a single hospital? Preferably in a public hospital?

    I am doing organizational level research on budgeting and budgetary control system in one public hospital, so i need good priori researches that has been conducted on the issue. Thank you in advance for your help.

    Gedion Firdawek · Arba Minch University

    Alright, Mr Azime, I am doing the research in an individual hospital, so regarding availability of data i am planning to conduct interview with the hospital's CEO and finance officer as well as other key informants in the hospital. I was also planning to distribute questionnaire to the clinician managers in the hospital concerning the budgetary process, but i realized that there is no departemental budget in the hospital. Therefore, the main tool of data collection in my case will be interview with CEO and finance director of the hospital. As far as secondary data in concerned, i will try to get the budgeted and actual income and expenditure of the hospital to do budget variance analysis.

    Regarding the methodology, A case study method is selected as the research method in the study which involves careful and complete observation of the budgeting system in the hospital.

    Also, a descriptive research or ex post facto research design will be carried out to clarify and define the nature of budget planning, and control system at the hospital. This is because the researcher has no control over the variables, but only reports what has actually happened or what is happening.

  • Mario Niepel added an answer in MTT Assay:
    Does anyone know the concentrations of Methotrexate for an LD50 in cancer cell lines?

    I am performing a treatment of HeLa cells with Methotrexate in order to find the LD50. I am using a range of concentrations from 4nM to 96nM. The treatment takes 4 days and I am counting the cells with the MTT assay. Does anyone have an idea?

    Mario Niepel · Harvard Medical School

    Michalis, drugs like Methotrexate are actually surprisingly complicated to work with. The reason being that folate in the media affects whether the cells respond to methotrexate. For example, if you increase the number of cells, decrease the amount of media, or lengthen the assay in all three cases methotrexate sensitivity increases. This makes it rather difficult to assess what the 'true' sensitivity of a given cell line is to methotrexate. Couple that with the fact that different cell lines have different media (with different folate or folate precursor concentrations), diffferent growth rates, and possibly different folate consumption, the whole thing can get really messy.

    That being said, I think the safest bet is to look what others have done and maybe stick to that. Alternatively, try to make sure that everything is available in abundance over the course of the assay (i.e. there is room to grow, plenty of media, and the assay is relatively short).

    Good luck.

  • Charles Francis added an answer in Theoretical Physics:
    Is the concept of field the proper way to describe physical processes?

    What is a field? Everywhere around a 'source' of the field, if we put a 'test particle', then a 'force' will be applied to it, coming from the source.
    This definition has a critical drawback:

    • if you calculate, using volume integrals, the total energy of the field , then you will find it infinite. Is this acceptable?

    On the contrary, there exists the interaction from a distance view, where a force is applied to the test particle only when it is being present in the neighborhood of the source. So, we have not any kind of infinite energy here.
    Which approach do you believe that is correct and why? 

    Charles Francis · Jesus College, Cambridge

    No, Akira. You are talking nonsense as usual. This is a simple integration. I am not insulting you. You insult science and display dishonesty with your accusations. If you were honest, or capable of coherent thought, you would have learned how to do this integration before posting nonsense and making slanderous accusations of corruption based only on the fact that you have no maths or science education. 

    Now, Akira, I have given you a scientific assessment of your accusations, and I have answered many of your questions, like pointing out that in the cloud chamber uncertainty in position is over the whole volume of the track. It is not my job to teach you physics 101 or high school calculus. I suggest you find a teacher and pay for lessons. This is supposed to be a forum for researchers, not a kindergarten for idiots and trolls.

  • Angel Jurado asked a question in Scientific Publishing:
    Has anyone used encyclopedias to look up for info in the Chemical Engineering/Organic Chemistry Fields?? Do you have comments on that?

    Hi Everyone

    I’m currently working on a summer project to try to understand the best ways to upgrade the user experience of one of the biggest scientific publishers in the world. I’m particularly interested in understanding how scientists interact with publications and encyclopedias in organic chemistry/chemical engineer fields, so I can give them some advice on how to best improve accessibility and usability of the scientific content they put out there. I would truly appreciate your insights on this matter and I’m seeking researchers that currently work in R&D departments of Pharma or Chemical organizations in the US, UK, Germany, France or Japan.  We will pay you for a 1-hour telephone interview, and your identity is kept confidential.  If you would like to participate in this initiative, please contact me through private message or send me and email to angel.jurado@rutgers.edu

  • Tomofumi Miyashita added an answer in Materials Science:
    Are there any leakage currents using doped Ceria electrolytes in SOFCs?

    Using YSZ electrolytes, the open circuit voltage is 1.15 V.

    Using doped Ceria electrolytes, the open circuit voltage is only 0.80 V.

    They said that there should be not only RI-drop, but also polarization voltage losses.

    However, every electrode degrades with time.

    Consequently, polarization voltage losses should become large and the open circuit voltage should become lower with time during electrode degradations.

    But experimentally, the open circuit voltage was never changed during electrode degradations.

    Please make sure by yourself.

    Necessity of verification of leakage currents using Sm doped Ceria electrolytes in SOFCs

    Journal of Materials Science (Impact Factor: 2.31). 01/2006; 41:3183. DOI: 10.1007/s10853-006-6371-8

    https://www.researchgate.net/publication/52013024_Necessity_of_verification_of_leakage_currents_using_Sm_doped_Ceria_electrolytes_in_SOFCs

    Tomofumi Miyashita

    Prof. Peter Vang Hendriksen must make sure by himself.

    I want to talk about this topic still yet.

  • Ani Ioana Cotar added an answer in rRNA:
    Can PCR product be used for template in PCR reaction?

    I have done PCR of 16S rRNA from bacteria genome. I used 27F and 1492R primer for the PCR. The concentration of the PCR product is small, so I want to repeat again the PCR with PCR product before as the template. Could it?

    Ani Ioana Cotar · Cantacuzino Institute

    Yes, you can use your PCR products as template in another PCR test using the same primers to increase the concentration of your PCR products

  • Ralph Eichenberger added an answer in Landsat:
    Can someone give a comment on the following topic - aspect/slope corrections?

    Aspect/slope corrections for Landsat data should be made after conversion to surface reflectance or should be done before that ? I'd like to use data from different sites across mountain  areas for classification of reflectance values. I was wondering if getting Landsat SR data from USGS can be processed for aspect/slope in order to get better training sets.

    Ralph Eichenberger · None

    I think it might work. With a little patience. Kind Regards

  • Sarang Mahajan added an answer in Bacteria:
    What is the result of induction passed a log phase of bacterial growth?

    Normally we will start induce our protein after an OD of the bacteria reaches 0.6 to 0.8.  What will be happened to our protein if we induce after the bacterial OD about 2 at 600 nm?

    Sarang Mahajan · Indian Institute Of Science Education and Research, Thiruvananthapuram

    0.6 to 0.8 is the OD when bacteria is in exponantial phase of the growth.

    If you induce very late you will not get a good concentration of your recombinant protein. On the other hand if you induce very early, due to high concentration of recombinant protein bacteria might turn into inclusion body.

  • Minerva Singh added an answer in Lidar:
    How might I calculate foliage height diversity (FHD) using multi-return LiDAR data? Can I use height percentiles like p01,p05,p99 etc for this?

    This is one paper that provides the the classical FHD formula ( -∑ pi loge pi)  and recommends using proportion of horizontal vegetation

    (http://www.isprs.org/proceedings/XXXVI/8-W2/HASHIMOTO.pdf).

    Others recommend using the Shannon index

    (https://www.researchgate.net/publication/222230782_The_use_of_airborne_lidar_to_assess_avian_species_diversity_density_and_occurrence_in_a_pineaspen_forest).

    I am confused as to which approach to use. Further I have height percentiles (from LasTools). Is there any way of using these to calculate FHD?

    Minerva Singh · University of Cambridge

    Hi

    @Shital: Bical is a great tool but it gives errors

    @Monica where in las canopy can I find the option for generating the height bins. Do you have a screenshot I could use? 

  • Md Moniruzzaman added an answer in Plant Analysis:
    Is it -LC/MS - the most effective and helpful for plant analysis?

    Especially for aqueous extracts

    Md Moniruzzaman · Dongguk University

    If you perform HPLC-MS analysis then it would be more better.

  • Khalid Eldahan added an answer in Antibody Generation:
    Inquire about blocking non specific sites in IHC?

    I work with rats doing many immunohistochemical reactions, I have seen that instead of using BSA as blocking reagent, sometimes is recomended that we use the serum of the animal in which the secondary antibody was produced. However wouldn´t be easier if we used a secondary antibody that was produced in rat (in case you use rat tissue) against the species where aur primary antibody was generated?

    Khalid Eldahan · University of Kentucky

    I also do a lot of IHC in rat tissues. We have a nice working protocol that uses serum from the same species as the secondary antibody species. For example, if I'm using Goat-anti-mouse secondary, I include 5% normal goat serum in the buffer. I would also recommend pre-adsorbing your primary antibody 1:1 normal rat serum. This also helps eliminate non-specific interactions of your primary antibody. We also make our own normal rat serum in-house. Much cheaper than purchasing!

    Best,

    KE

  • Francisco Murphy Perez added an answer in Free Radical Biochemistry:
    Can the solvated electron be classified as a (free) radical?

    The IUPAC gold book says a radical is:

    "A molecular entity such as .CH3 , .SnH3 , Cl. possessing an unpaired electron."

    The only document where someone "prefer(s) to regard the simplest free radical as an electron" is here http://pac.iupac.org/publications/pac/pdf/1967/pdf/1501x0015.pdf
    (paragraph 2, line 12).

    Also from the IUPAC, although a little old.

    Any light on this would be greatly appreciated.

    Thanks

    Francisco Murphy Perez · Universidad Nacional Autónoma de México

    I name it like so in my thesis and my advisor did not like it.
    I know is just a name ...

  • Khristin Fabian added an answer in Appropriate Technology:
    How do you feel about lecturers being allowed not to use technology effectively?

    Our students are inhabitants of the technology age, therefore I believe we have to use technology effectively to support teaching and learning. Personally I do not understand how a lecturer can be allowed to print an assignment to mark it using a red pen - if the students have submitted documents created in Word, Excell or Powerpoint. By using track-changes and comments, a track record can be created to support the students in revising their assignments. According to me, this is absolutely needed when we are assessing the chapters submitted by M and D students. I believe the use of technology should be integrated as an essential aspect of the assessment of the work of lecturers. Furthermore, I also believe that such lecturers would not be able to move into the e-learning industry. I would like to hear how you feel about such reluctance to update skills to improve practices.

    Khristin Fabian · University of Dundee

    It is interesting to see the answers here but it is important to note that whilst we may all agree about the importance of lecturers updating their skills (and i do agree that its important but I do have other ideas about what technology skill is important), there's already some bias here beginning with the participants answering this question. you might end up with everyone in agreement but that everyone is part of a group involved in social media such as this. 

  • Raluca-Maria Bălă added an answer in Cointegration:
    How do I treat stationarity with trend in ADF test in my model?

    I have performed ADF test for a 4 variables I am considering for multivariate analysis. The ADF test at the log levels of the data are all insignificant. After taking the first log difference, all the variables with their t-statistic are significant considering constant and constant with trend except one (particularly exchange rate data) which is insignificant with the constant only but significant with the constant and trend ADF model.

    Obviously its graph somehow shows trend at level. Now

    1. Can I consider the data at the first log difference as stationary using the trend or not stationary using the constant only. I wish its stationary as the other 3 variables so that it pass the test for my cointegration analysis I want.

    2. If I consider it as stationary using trend ADF model, how do I treat this trend in the actual model. Inclusion in the model or the cointegration model.

    3. If I dont consider this data as stationary at the first log difference with trend, it means somehow it is I(2), and sure after taking second log difference makes it stationary. But not what I want for cointegration analysis.

    What do I do. Hope someone can help me here. 

    Raluca-Maria Bălă · Bucharest Academy of Economic Studies

    Emmanuel, as you also said before, the small sample size and /or a structural break in the series will be a problem in obtaining reliable results in unit root tests such as ADF, PP or KPSS. So, taking these into account you should see if your series are indeed I(1) to be able to continue with your cointegration analysis. Nevertheless, as John c Frain said the exchange rate series should be a random walk, that is stationary and you could not use it in the usual cointegration tests. Hope this helps.

  • Inbesat Khan asked a question in Cell Line:
    Difference Between MOLT-3 & MOLT-4 cell lines ?

    although the details about these cell lines are given on ATCC website but i thinks there is very minor difference between the two . please enlighten me with your answers.Thank You.

  • Lily Goldstein added an answer in Aseptic Techniques:
    Can anyone tell me what this stuff is in my primary neuronal cell cultures? I have pictures! Contamination? Weird cell growth?

    I culture primary neurons, with conditions that support neuron growth over glia or astrocytes. This problem began completely out of the blue, with no changes to protocol, aseptic technique, media or cell source. We've noticed a problem in the inducibility of activity using Bicucullin A, we’re only getting about 10% of the induction that we used to, at both the protein and mRNA level (sometimes no induction at all). Contamination seems unlikely at this point, as we've tried culturing on plates that support bacterial growth and a wide variety of antibiotics. This is a recurring problem, not just an isolated incident, it occurs each week with a new animal source. The contamination/whatever-it-is does not show up in plates that have been prepared and stored identically but have no primary cells.

    I've attached a picture at 20x of the culture after 12 days of growth and a culture after 6 days of growth (with far fewer cells plated than normal). The weird orange-yellow stuff is what I suspect is contamination (or maybe a really unhealthy neuron?) Has anyone run into anything like this before? Any suggestions at all?

    Lily Goldstein · University of Massachusetts Boston

    I attached another picture at an earlier day, with about half as many cells plated. I think there are way less dead cells (bright white blobs, as seen in the day 12 culture), but still a fair amount of the orangey yellow stuff that we think is affecting inducible expression levels.

  • Max Stanley Chartrand added an answer in Medicine:
    Does the responsibility of researchers end with the scientific publication of their findings?
    Or should he or she also ensure that these findings find a way to a) non-scientific public and b) the implementing authorities / institutions (a practical reference provided)? I have made it my habit to any scientific contribution to compose another layman's contribution and to publish in order to create the possibility of practical implementation. All non-academic partners are extremely grateful for it. Without access to databases they would probably know nothing about these results and findings. Other ways to make research applicable?
    Max Stanley Chartrand · DigiCare Behavioral Research

    True, and this is particularly true of research that is designed to answer a very narrow, short term question only to present its conclusions as if the results can be broadly applied over a long time span. Monied interests, which drive the majority of research in the world, have a tendency of dictating outcomes to those charged with finding them.

  • Timothy Bahry asked a question in Tracking:
    Can accidents be reduced by creating trains with low friction wheels and rails?

    That way, if the train starts moving while somebody is on one of the rails under it, the wheel that first touches them will push them along the rail instead of cutting them up. If in addition to that, the surface under the tracks is a low friction ultra smooth substance instead of Cement like it is for the ICE train in Germany, the person caught by the train wheel won't lose a lot of blood from being scraped by the ground under the tracks either, giving more time to notify the train company of the problem to stop the train.

  • Norbert Bittner added an answer in Pyrogallol:
    Can an increased level of hydrogen peroxide reverse the reaction of superoxide dismutase in an assay ?

    I'm having the problem that in my SOD assay (NBT assay, Pyrogallol assay) the sample reaction is showing a faster O2- production than the blank of the SOD standards. Means that the samples are way darker in color after 5min of reaction than the SOD blank. I'm working with Sephadex desalted pine crude extracts.

    Norbert Bittner · Freie Universität Berlin

    @ luis  thanks for the quick reply.. I had the problems without sephadex and without PVPP as well, i thought this would solve it by catching phenolics with PVPP and Sephadex desalting to get rid of low molecular stuff.. And nbt is not stimulating the O2- production it is the phenazine methosulfate (PMS) that does it.. For the SOD standards. I do a dilution series and they work well. So the assay is okay it is something in the extracts..

  • Nai-Kei Wong added an answer in Cell Imaging:
    What kind of cell line can I choose in cell -imaging for caspase-4 activated by TLR-4 ligand?

    I want to do the cell imaging of caspase-4 that activated by the TLR-4 ligand. However, I do not know , which kind of cell line that I should choose. I hope someone can tell me . Thanks very much.

    Nai-Kei Wong · The University of Hong Kong

    If species is not much an important concern, I think RAW264.7 (mouse monocytic macrophages) are certainly much more responsive to TLR4 ligands, compared to THP-1. 

    I used 5 ng/mL PMA to differentiate THP-1 cells (3-5 days, depending on needs). But I never used differentiated THP-1 cells to detailedly study LPS response. 

    In addition, even with PMA-differentiated THP-1 cells, strictly speaking, they can be further polarized to true M1 or M2 cells. These preparation requirements make THP-1 a rather expensive model. 

    See:

    http://www.nature.com/onc/journal/v31/n14/fig_tab/onc2011370ft.html

    http://www.nature.com/jid/journal/v129/n4/fig_tab/jid2008310f2.html

    ***

    In low passage number RAW264.7 cells, LPS (or LPS+IFNg) response is obvious in overnight treatment (>12 h) and easily reproducible. 

    Primary mouse macrophages (BMDM) differentiated with GM-CSF (1 week) behave quite like RAW264.7 cells, in my experience (but this depends on what phenomena we are talking about). 

  • Luca Puviani asked a question in Acquisitions:
    In a Multiple Separately stimulus conditioning scenario and further extinction of only one of them. Will the others pre-acquired CSs change?

    In Classical Conditioning one perform a CS1->UCS acquisition, then separately a CS2->UCS acquisition (the UCS must be the same). Let's suppose to neglect the context cue, or let's assume the CS1 consists of both a cue1 and a context1 and the same for CS2 which consists of a different cue2 and a different context2 respect CS1.

    Finally follows the extinction of only one of the two, let's say CS2. Does CS1 change respect the CS2-pre-extinction and CS2-post-extinction? There exists literature which details experimentally about this paradigm?

    Luca P.

  • Ramón Piloto-Rodríguez added an answer in Octanes:
    What is the effect of adding fuel of octane number 95 with fuel of octane number 80 in the fuel tank?

    What is the effect of adding fuel of octane number 95 with fuel of octane number 80 in the fuel tank

    Ramón Piloto-Rodríguez · Instituto Superior Politécnico José Antonio Echeverría

    The effect is an average (not accurate) of both octane numbers that will also depends on the amount in volume that you will add to the blend; therefore the calculated average is dependent on the relative percentage. Anyway, the difference between the calculate number and the real number established by engine tests won’t be far. A good mixing of components is important.

  • Joaquim Antonio Mourato added an answer in Social Responsibility:
    Do you have or know studies about social responsibility in higher education institutions?

    Polytechnic Institute of Portalegre has implemented a social responsability system. It´s only institution in Portugal with this system. So it´s important for us find other examples.

    Joaquim Antonio Mourato · Instituto Politécnico de Portalegre

    Thanks for your contribution.

    Best regards

    Joaquim Mourato

  • George E. Van Hoesen added an answer in Theoretical Physics:
    May gravito-electromagnetism be an alternative to Einstein's theory of general relativity?

    Gravito-electromagnetism (GEM) describes gravitation starting from the idea (suggested in 1893 by Oliver Heaviside and further developed by Olev Jefimenko) that there is a formal analogy between the gravitational and the electromagnetic phenomena.  This implies that - relative to an inertial reference system O - a gravitational field is characterized, in analogy with an electromagnetic field, by two vectorial quantities: the gravitational field Eg and the gravitational induction Bg.  The spatial mass destribution is the source of Eg  and the motion of the masses relative to O that of Bg

    According to GEM, a gravitational field is just like an electromagnetic field (Maxwell's equations) mathematically described by a set of four partial differential equations, the GEM-equations.   They describe how Eg  and Bg   vary in space due to their sources and how they are intertwined.  The action of a gravitational field on particles is described by a law analogous to Lorentz's force law: the force law of GEM.

    It has been shown that GEM can explain a number of phenomena (precession of planets and binary pulsars, gravity probe B experiment, ...) that are inexplicable in the context of Newton's description of gravitation what makes that GEM can be considered as a possible alternative to GRT.

    In line with the original idea of Heaviside GEM can be seen as an extension and a refinement of the "classical theory of the gravitational field", an extension that - besides the effect of the spatial distribution of the gravitating bodies - takes into account the effect of their motion.  GEM can theoretically be founded and explained by the theory of informatons. (https://www.researchgate.net/publication/268576021_GRAVITO-ELECTROMAGNETISM_EXPLAINED_BY_THE_THEORY_OF_INFORMATONS?ev=prf.pub)

    Let us still mention that GEM has been discussed within the context of general relativity by a number of authors and that it has been shown that  the GEM equations can be derived in the weak field approximation of Einstein's description of gravity.

    George E. Van Hoesen · Global Green Building LLC

    Akira,

    You are correct on so many levels.  There is so many misunderstandings in Physics today that I am not sure where to start.

    The Wave Particle problem can be looked at in a different way and I can get No one to even think in the same line that I know is correct.  A particle that is moving though space and time with any sort of spin, charge, angular momentum, or mass of any kind would have to look like a wave to the observer.  The faster it travels the harder it is to determine the mass is there.  The speed of light masks the mass but does not mean it is not there.  We have realized this with the Neutrino but not with the photon yet.  

    We have in physics more than 100 years ago given up on the idea that the duality that appears to be there can be explained by moving a particle through space with rotation and or charge.  As a matter of fact the faster the particle the more spread out the affect it has on things it moves past.  

    The conclusion that I came to decades ago is that there is no Wave Particle Duality...  It is a particle that has wave like properties.  Its motion and its ability to effect other mass is because it has motion through the mass at light speed.  The disturbance is equal on either side but in opposite direction and therefor overall a net zero.