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biophysics, protein folding and stability, spectroscopy, bioinformatics, molecular dynamics. . . .
Questions related to Biophysics
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The phrase in the Title line imitates Karl Popper’s All Life is Problem Solving.
Since thermodynamics plays a role in life processes, it was surprising that searching “All life is thermodynamics” on Google on August 16, 2022 gave no results.
Don’t organisms seek to maximize and preserve their entropy? And to optimize their use of energy and outcomes based on energy inputs? Aren’t survival and procreation ways of preserving previous products of energy use?
Is there justification for the statement, All life is thermodynamics? Or is the statement too simple to convey any insight?
Schrodinger in What is Life referred to thermodynamics, statistical mechanics; chapter 6 is Order, Disorder and Entropy. And more recently there is: J. Chem. Phys. 139, 121923 (2013); doi: 10.1063/1.4818538 Statistical physics of self-replication by Jeremy England.
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Hello Robert; Here are a couple of aphorisms that relate to your question;
1. Life is an entropy-retarding process.
2. Organisms traffic in energy.
These observations reflect the fact that maintaining biomass requires ongoing access to potential energy. They are an expression of the laws of thermodynamics. That was fun to think about. Best regards, Jim Des Lauriers
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What are the Biophysical and Biochemical techniques that used in Recombinat DNA technology??
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Biochemical methods are based on specific reagents and chemical transformations, using e.g. enzymes, antibodies, ligands etc
Biophysical methods are based on equipment and spectral properties: absorbance and fluorescence spectroscopy, light, x-ray and neutron scattering, nuclear magnetic resonance, Forster resonance energy transfer
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One might argue: Animals increase their survivability by increasing the degrees of freedom available to them in interacting with their environment and other members of their species.
Right, wrong, or in between? Your views?
Are there articles discussing this?
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Also check please the following useful RG link:
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Does the DNA remain stable or degrade at this temperature? Would there be any difference in thermal stability between supercoiled and linear forms of say, 3 kb plasmid.
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Here`s the article `The conformation of double-stranded DNA` by B. Cyriax &  R. Gäth, Naturwissenschaften, v. 65, p.106–108 (1978).
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During AFM imaging, the tip does the raster scanning in xy-axes and deflects in z-axis due to the topographical changes on the surface being imaged. The height adjustments made by the piezo at every point on the surface during the scanning is recorded to reconstruct a 3D topographical image. How does the laser beam remain on the tip while the tip moves all over the surface? Isn't the optics static inside the scanner that is responsible for directing the laser beam onto the cantilever or does it move in sync with the tip? How is it that only the z-signal is affected due to the topography but the xy-signal of the QPD not affected by the movement of the tip?
or in other words, why is the QPD signal affected only due to the bending and twisting of the cantilever and not due to its translation?
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I agree with Annemarie Honegger.
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I am computing Van der Waal interactions in python for a peptide of size 10 residues for various conformations. The total conformations (or the number of PDB files is 300,000). Is it possible to compute only the 1-4 atom distances to compute Van der Waals interactions as the bonded and 1-3 atom distances are irrelevant when it comes to Van der Waal interactions using some python module?
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Hello,
I intend to perform thermal unfolding of my proteins by CD, to determine their melting temperatures.
I initially scanned the proteins over a wavelength range of 250 -190 nm at 25 °C (in 20 mM Phosphate bufffer pH 7.4) to determine their structural content. The results confirm that the proteins are made up of alpha-helices (which is what i expected), with two negative bands at ~208 nm and ~221 nm, and one positive band at ~ 192 nm . See attached image!
Next, I will like to thermally unfold the proteins at a specific wavelength. Given the results attached, could anyone please suggest what wavelength would be idle to monitor the thermal denaturation of the proteins and why?
Best
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The earlier mentioned 222 nm find its origin in the following source: The ellipticity at 222 nm has been used as a rough measure for the relative helicity, for which q222=36 300 deg.cm2/dmol was taken as 100% a-helix (Hodges et al., 1988).
Hodges, R. S., Semchuk, P. D., Taneja, A. K., Kay, C. M., Parker, J. M., & Mant, C. T. (1988). Protein design using model synthetic peptides. Peptide Research, 1(1), 19-30.
You can find the paper here:
and in Figure (they actually used 220 nm) examples of thermal melting profiles).
Best regards.
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Compiled allometric data might help to detect scaling patterns.
Or similarities in the scaling relationships might suggest connections otherwise too subtle to find.
Does such a list exist?
Does such a list exist for biological phenomena?
If such lists do not exist should they?
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No such compilation that I know of (only some reviews, for the scaling of a specific trait in a specific group). So regarding your last question "should it exist", I think creating such a list would be an admirable but difficult task - depends on what you're thinking of exactly as "all known instances". Using all the raw data available would basically be a "database of everything", virtually impossible. It would still take a ton of work to even make a list of every allometric equation ever explicitly stated for every trait in every organism, and you'd also need to be able to update it, and to subset it by trait, taxa (down to intraspecific resolution, don't forget), external factors such as region/temperature/season/age/sex, etc.
(btw if somebody does go and make such a database it should definitely be named ALLometric right?)
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I want to know if the number of fringes and their shape is an important factor for the accuracy of phase definition?
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Hello,
Our old but not to old DynaPro Plate reader I does not work anymore and Wyatt does not want to investigate the problem as the instrument is 10 years old. We have the money to pay them but they really do not want to loose time on it...
We would like to know if some of you know DLS instruments that are compatible with the measurement of several conditions (at least 30 conditions) in parallel. Of course the goal is to find a company that is able to do a maintenance.
Best,
Sébastien
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Protein aggregation over a range of temperatures (protein denaturation) is a common application for the plate reader. The wells can take tiny amounts of fluid. Have you spoken to Malvern SA?
Bâtiment Le Phénix 1, 24 Rue Émile Baudot, Palaiseau, 91120 France
Tel: Sales: + 33 1 69 35 18 08
Tel: Support: + 33 1 6935 1806
Fax: + 33 1 6019 1326
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If I use AVI, BSI, SI and TI as biophysical factor.
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Please see attached file
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Hi I have attached the link to article and the table from the article with this question, I wanted to know how the value of 0.7 s-1 is calculated from the slope and intercept values using the bell evans equation. The values of slope is 85.3 and intercept is -351.
Thank you in advance
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0.048/4.1*exp(-((-351*0.048)/4.1))
kBT = 4.1 pN*nm
It is clearly written in equation 5 of that paper.
However, I would advise using better models for such data analyses: https://iopscience.iop.org/article/10.1088/1742-5468/ab6a05/meta
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I have a set of Ramachandran angles. I wish to make Ramachandran plot out of it with standard allowed region contours in the background in Python. I couldn't do that in python. If not in python, Is there in anyother software where I can do this?
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Is anyone aware of a scientific answer, specifically what type of biochemical or biophysical triggers can trigger activation?
Thanks.
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endogenous retrovirus comprises up to 5–8% of the human genome, it is vertically inherited.
The majority of ERVs that occur in vertebrate genomes are ancient, inactivated by mutation, and have reached genetic fixation in their host species. For these reasons, they are extremely unlikely to have negative effects on their hosts except under unusual circumstances such as (people with schizophrenia), therefore we can suggest that some Neurological disorders lead to activation ERV
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Biophysical remote sensing pertains to the detection and analysis of the biological and/or physical characteristics of a landscape, particularly its vegetation and soil.
In carrying out a research in biophysical remote sensing and it's application to climate and land change science what methods or approaches can be used?
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The easiest way to analyze the biological properties of the landscape is to use spectral data in GIS programs like QGIS, ENVI, and ArcGis. Also you can search pre-prepared data at https://earthengine.google.com/
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This question leads me to ask another one.
Why do the osmoles of urea when they pass into the cell exert an osmotic effect and draw water into the cell, while when they were outside they had no effect?
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Urea freely crosses the cell membrane, therefore independent of the osmolarity of the solution, it cannot build up a gradient of osmotic pressure across the cell membrane - The extracellular urea concentration will quickly equilibrate and build up a similar intracellular concentration.
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Hi! I am quite curious about mitochondrial mechanobiology. How does mechanotransduction (both cell-matrix and cell-cell) affect mitochondial behaviour (mitophagy, mitochondriokinesis & ?) What is the signalling pathway behind (both transcriptomic/proteomic and metabonomic)? How are mitochondrial behaviours and signalling in wound-healing process? Are there some most nonnegligible/core article in this topic?
Thank you!
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Very interesting topic that motivates the search
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Hi,
I and working on a project for extraterrestrial life, and i need few work on the titled topic. If is there any data, recommend it or please discuss the evaluation mechanism.
Thank you,
Muhammad Furqan Ali
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You can search for the following articles: National Environmental and Natural Resources Information System Environment and Temperature Report, light, atmosphere, wind
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Hi everyone
Could you share your research and/ or other researches related to the application of magnetic fields in biophysics and Medical physics?
Thanks
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Also, one can mention this although rather exotic technique https://en.wikipedia.org/wiki/Magnetoencephalography
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I need a way to rotate my entire system (DNA and solvent) such that the axis of the DNA lies along the x-axis, but I want the periodic boundaries to be preserved. I added pictures of my system before and after rotation.
How can I edit things in such a way that the entire system is rotated, but molecules stay within the box? (basically the water molecules that were rotated down and went outside the box should reappear up inside the box)
Thank you in advance!
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PBCs are always respected from a simulation perspective as far as you have turned on them in the .mdp file.
If you want a rotated system for the purpose of having nice images, then play around with gmx editconf -rotate/center/translate and when you have the orientation you want clean up the PBC representation by shifting everything inside the box with gmx trjconv -pbc mol or similar. Adding the flag -center and selecting the DNA could also be helpful in this case.
Hope this helps
Nicola
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Dear Sirs,
I did not find this material on the internet. There are only mechanical models of some aspects of self-replication. Full mechanical model is absent. Of course it is enourmous problem if one precisely build it. But maybe there are simple and simultaneously more complete mechanical models? I prefer purely mechanical self-replicating machine but self-replicating robots are also good.
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The first mechanistic model of self-replication was given by John von Neumann by his self-replicating cellular automaton. He was followed by others: Langdon, Reggia, ...
It would be interesting to study this research stream as it provides great insights into creation of mechanistic description of certain properties of living structures.
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How can/should we distinguish between a Biochemical and a Chemical Reaction.
As per one explanation:-
Chemical reactions are discrete reactions with catalyst involved in the process where as in biochemical reactions there are a series of reactions involved with the product of one acting as the substrate for another and this complex process of interchanges taking place with the involvement of enzymes.
For a more specific example if we are conducting photosynthesis in vitro then it will be considered as a biochemical reaction rather than a chemical reaction.
But the dilemma stems from the fact that, even chemical reactions go through complex series of steps, like any organic synthesis reaction. In this case also there is the involvement of catalysts like enzymes. Thus, can we consider it as a biochemical reaction! But mostly we only attribute it to be a chemical reaction which is indeed the case.
So, what is the proper difference or point of distinction between a biochemical and a chemical reaction. How can we exactly relate that one reaction is a biochemical and the other is chemical!
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Dear @Mrutyunjaya Panda All biochemical reactions are chemical reactions. I agree with Dr @Frank T. Edelmann in that in principle, there is no major difference. The same reaction can occur 'in vivo' and 'ex vivo'. You can also access a similar discussion at the following link:
Best wishes, AKC
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Looking for a sequence or vector map of pEC86, a plasmid which expresses E. coli cytochrome maturation (ccm) genes.
Here is a reference for the plasmid:
Arslan, Engin, et al. "Overproduction of theBradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase inEscherichia coli." Biochemical and biophysical research communications 251.3 (1998): 744-747.
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Hello
Did you ever find the sequence of this vector?
If so, would highly appreciate it if you could share
Thanks
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I need brief information about the field of the researchers who work with bioimaging techniques in their researches. In Finland, there is the public concept that they think a researcher with biomolecular and biochemical background must stay in his/her old methods of researches which is work in the lab and pipetting, etc. I am so much interested to know is it true about all scientific groups that you cant get a chance of trying new techniques and also learning which suppose to be the main purpose of science and its progress or as I understood it is just an excuse? Science cant grows with a limit or making exclusiveness.
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Breakthroughs and discoveries in science usually occur when scientists do something different or notice something odd. This happens far more frequently on the boundaries between disciplines than in the core of the discipline. For example, sequencing DNA used to be a very slow, painstaking process. Then the instrument makers got involved in the biochemistry processes and things got much quicker, paving the way to more discoveries. The fun science is usually the science which combines multiple fields, so learn and do what you enjoy. You will be far more successful than if you artificially limit yourself. But beware of overcommitting, as well. Have a goal and do what it takes to get there, but avoid distractions as much as possible. Finally, be aware that what you study in college does not necessarily define your career. I am a physicist by training, but worked for 15 years as a professional computer programmer. I have had no formal computer programming instruction, but learned it all by doing. Just keep learning and doing what you love, and you will never run out of things to do. Good luck!
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For the simulation purpose, I need force-field for Mn3O4. I searched and tried a lot but almost unable to get appropriate force-field parameters for it. Can anyone help me by suggesting or availing it ?
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You can try with Swissparam to generate required force-fields parameters.
Please follow the link:
Upload your structure in .mol2 format. You can use Avogadro or Jmol to prepare the structure in .mol2 format. Once prepared, run the .mol2 structure and wait for few minutes to get output file from Swiss param based software and then do the required changes for the force-field parameters.
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What impacts do these drivers have on socioeconomic and biophysical activities in the country
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In a small search, the climate of South Africa is cataloged with mild and rainy winters, and hot and sunny summers, however the atmospheric effects will sometimes show a significant deterioration.
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Which location of atoms is being called a Phi=0 degrees?
Which location of atoms is being called Phi= +ve? (such as +90 degrees)
Which location of atoms is being called Phi= -ve? (such as -90 degrees)?
Same question for Psi.
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If I can I will answer. Proteins need parameters, notes, numbers, notations to simply characterise them. In a very similar manner to organic chemistry in bonding, they describe several properties which describe how the b9nds are in a protein. The torsion and the tension phi and psi. They describe up to which point the atoms in a protein separate or "twist" in space. This means each protein can be described by its angles up to the point of placing them on a chart (Ramachandran). This means the proteins can be grouped by its "topology". The numbers mean how much the aminoacids are laid to form the protein (at least the tertiary conformation, but it may mean secondary). What I am not clear if the proteins cannot exceed 180º. (Is this the limit?) when you mean the numbers 0-90-0 or 90-0-0 which is a two way graph (normally, maybe for tertiary there are more axes) does this mean I am reaching those 180º? If I had to say, it would be alpha conformation and beta conformation. This can be found on a graph. The extreme right (and maybe on the upper side on the lower side it does not exist or it is not found on Earth, maybe be coil-coil conformation). 90-0-0 is alpha and 0-0-(-90) is beta. 90-0-0 would be coil-coil and not defined as a protein. It would have to be an extreme rare protein. I would need to check the updates or if it means anything with another axes. Maybe I need to check a Ramachadran graph and the notation on the angles, but it would be more or less on thee guidelines...
Thank you (sorry). Please come and write again if I have not been clear
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Dear all,
Besides biophysical or Biochemical approach, is there any other way of studying protein-protein interaction. I am planning to work on viral protein interaction with host proteins. The conventional method to study is yeast hybrid tool. However, I would like to explore other approach. Please suggest the way other that yeast as it takes huge amount of time and mutations issues are always a concern in yeast that results into some unexpected unexplained results. looking forward to hear from you.
Thank you in advance
Prem
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Thank you very much for sharing the posts
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What are the differences between Medical and Biophysics?
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1. biophysics is the study of biological processes and materials through the theories and tools of physics; the application of physical methods to analyze biologic problems and processes.
2. The study of physical processes (for example, electricity, luminescence) occurring in organisms.
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I'm taking some measurements using Raman spectroscopy of healthy pig eye samples using a new excitation wavelength and my supervisor said to choose a sample size to allow for spec/sens of 85%. These measurements will be identical just on different, healthy eye tissue to simply show that I obtain similar things each time. Is there a way to estimate/calculate what my sample size should be for this or do I need previous data, numerical limits etc? Any help would be great, thanks!
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Based on the results that we have presented, a sample of minimum 300 subjects is often sufficiently large to evaluate both sensitivity and specificity of most screening or diagnostic tests
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I have had unheard of success with protein crystallography lately from a super successful protein expression and purification batch.
I have attained a lately reproducible vast amount of crystals of no average size. Is there a way to tell based on appearance which of these crystals should diffract the best?
All these crystals grow from a clear droplet in 12 hours.
What's the most important parameter?
* transparency?
* size?
* how intensely it reflects light? birefringence?
* geometry?
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Hi there, Unfortunately there is absolutely no link between the size and appearance of a crystal and its ability to diffract. Gorgeous crystals may diffract very badly and little dirty bits may result in structure at 0.8A resolution (The opposite may also apply!).
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It is well known that heating can denature proteins. However, what does happen to proteins in the case of short and ultrashort (microsecond or nanosecond scale) heating to extreme temperatures (100-1000 degrees of Celsius) ? Such heating occurs for example when applying ultra/short laser pulses or pulsed intense electric fields.
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At these short times proteins can withstand much higher temperatures. For example, gelatin gel (5-10 %w/v) remains solid when heated to 500-700 degrees C by IR radiation of microsecond duration. Instead, explosive evaporation of water will occur with mechanical damage to the tissue, possibly breakdown plasma cascades. The paper below LITERALLY has it all. Also you may read about Arrhenius integral which is a measure of thermal damage to macromolecules.
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Solving the protein structure prediction problem by AlphaFold2(AF2) from sequence seems at its core a game-changing breakthrough. Major areas of biology and biophysics will thrive and others may become diminished or even obsolete.
Will AF2 hurt or benefit experimental methods for determination of protein structure? Structures of large macromolecular machines should be enabled by having accurate computational structures for subunits and components. X-ray structure value may become more specialized. One thing that seems likely is that the already great value of sequence data (which is doubling every 8 months) is likely to become far greater by being more directly connected to spatial information. At its core solving the protein folding problem will enhance sequence impact and thereby increase the overall the pace of biology and biophysical advances by improving the ability of structural biology to better harness the flood of sequence data. How much will medical areas such as cancer biology and cancer drug discovery benefit? How about research areas such as protein design? What areas are likely to be most powerfully advanced and which most negatively impacted? What do you think?
How should senior and early stage researchers position themselves to ride the wave of growing positive impacts and reduce research wipeouts when this new mega-wave adds constructively or destructively with current research waves of systems biology and single molecule and single cell advances? What should we change in training for graduate students and fellows to insure they are correctly positioned for science with reliable protein structure prediction?
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John,
You have asked an interesting question. For a senior member of structural biology community it shows the yearning for practical solutions as well as naivety of the real difficulties in even defining the problem. The protein folding problem is an ill-defined and most likely unsolvable problem in its classic sense of axiomatic reductionist sciences. If the old Anfinsen view was true about the minimum of global free energy there never would be life on Earth or anywhere else. The new paradigm that I partially formulated is that every protein (as a matter of fact every macromolecule of life) has its own specific recipe how to combine the structural with dynamical features to accomplish the desired function (PNAS 106(2009)10505). It means that there is a unique proportion of conditionally stable structure to conditionally unstable elements to perform given function. This ill defined condition is not solvable by classic axioms but it just might be partially solvable by fuzzy methods such as AI. Why AI is so good? Because the protein folding problem is a practical not a theoretical problem. Depending on conditions and their possible changes the protein suppose to perform a certain function. This is what physics call "self organized criticality". This is far from equivalency of having a certain structure nor a possible unique binding site nor anything that remotely can be called a solution. AI just simply captures better the set of heuristic rules than other methods particularly dynamical coupling with solvent. There are millions of examples to support this view. If anybody wants to engage I will be happy to, but not now. There is no place here to mention even a single one.
So the only hope is that we found an effective heuristic tool that gives us better approximation to reality. To be properly tested the method needs to be exposed to finding bordering cases when it breaks down. What conditions produce divergencies. What percentages of certain structures are folded and are not. For instance only around 30% of genomes of higher eukaryotes are properly folded. Around 30% is conditionally folded (on the target, in the particular compartment) since the misfolding diseases. And finally around 30% is almost never folded with residual presence of folding nuclei that precipitate the function. So all these classes of proteins must be tested against this new AI approach to see when the rules break down and how to enhance them.
Therefore, it is not a breakthrough that John is so excited about. But it is definitely progress. But, I would not see anytime in the future, abolishing or significant diminished importance of any particular field of science, in exactly the same way as radio did not get extinct by TV.
Bog
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AF2 may most strongly impact experimental determination of protein structure by multiple methods with possible benefits and negative impacts over time.
Some areas may need to quickly pivot or become obsolescent, whereas other may thrive.
Structures of large macromolecular machines should be enabled by having accurate computational structures for subunits and components.
The already great value of sequence data (which is doubling every 8 months) is likely to become far greater by being more directly connected to spatial information.
Overall the pace of biology and biophysical advances can be expected in increase by the ability to better harness the flood of sequence data.
What do you think?
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X-ray people might be impacted more than NMR and cryo-EM people by AF2. It may replace all biophysical techniques for structural determination someday, IMHO, they advertise much better than academic groups for the things they've achieved and still a long way to go (like when Rosetta first introduced). Not to mention the missing pieces like non-natural amino acids, binding partners, dynamics, different circumstances in the cell, more or less scientists would like to validate the prediction experimentally. However, it is hard to argue with its valuable inputs and insights before wet bench works that cost a lot.
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Hey scientists,
I am intending to observe the extension of a short 10-mer primer on a 16-mer template. I just use a certain DNA polymerase to extend the primer on a templating strand with a matching or mismatching deoxynucleotide triphosphate (dNTP) and the necessary cofactor magnesium.
I was thinking that depending upon the polymerization efficiency, I might have products that are size "n", "n + 1", "n + 2", "n + 3", " n + 4", "n + 5", "n + 6" in size where size "n" is the size of dsDNA duplex without addition of dNTPs, or in other words no extension.
I was envisioning I just combine in a test tube the enzyme with the template:primer construct + the dNTPs of interest + MgCl2 and let the reaction happen for 30 minutes. Then I would denature my protein with heating the test tube to 50°C. (My polymerase isn't one of those thermal stable polymerases), and slow cool the test tube to room temperature.
I could run the products on an ethidium bromide stained gel with a control being a lane with just the template:primer construct with no extension, and then lanes with attempted extension of the dsDNA construct. If at least one base is added I want there to be a notable shift.
What resolution gel could achieve that?
4% Agarose or 16% polyacrylamide or something?
It would be cool, but it's not a big deal for me to distinguish exactly how many bases were extended from "n". So long as I can see an extension from "n" and I would be satisfied.
Thanks!
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I would use 20% denaturing PAGE on a long gel ( 40cm old fashioned sequencing gel rig) if possible and silver stain for visualisation. If you have access to a dna sequencer then you could put a long (20-30 base) 5' tail on your oligo to increase its size and dye label the oligo. Then if you run the extension products in the linear acrylamide capillaries it will give you single base discrimination. Size standards are a problem with short sequences which is why I suggest a tailed oligo to bring the product size into the range of the size standards
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There is a growing interest in developing means of early detection of crop nutrient deficiencies. It has held that by the time a deficiency shows up in a soil sample, the crop is already under stress. Does crop sap analysis help to resolve this information gap? If so, how can we expand the use of this from high margin specialty crops to commodity crops?
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Leaf and petiole analysis is an established tool. Protocol for perennial crops have been standardized for nutrient analysis.
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So I am trying to crystallize a Protein:DNA complex with DNA at at least 5 times excess at basic pH with a certain poleythene glycol molecular weight (PEG) and a mixture of soluble salts at low concentration less than 110 mM. I was getting a microcrystalline shower at a certain concentration of the components but lots of amorphous brown precipitate.
So I tried glycerol concentrations logically from 0 to 5%. I blocked the microcrystalline shower at glycerol concentrations greater than 1%. I kept increasing the concentration of glycerol regardless and at glycerol concentrations higher than 5% (v/v) and all other components in the system no brown amorphous precipitate appears; total protein is soluble. At glycerol concentrations greater than or equal to 15% (v/v) all other components in the system, the brown amorphous precipitate comes back. When glycerol is @ 13% (v/v) I got some small crystals not a showers worth. But nucleation seemed to fall asleep and that was it.
Then I lowered the concentration of one of the salts potassium citrate monohydrate to 100 mM and I get a reproducible white haze or white sheen appear over the droplet from the edge. The haze now seems to appear at any concentration of PEG much less than or equal to the concentration of PEG that I would expect to give me crystals. I also think the haze is growing.
I have seen this haze before when I crystallized the free protein under totally different conditions with different components in the system and acidic pH. This white haze would move from the edge of the droplet towards the center and then medium sized to large crystals would appear out of the haze in less than 30 minutes. Those crystals diffracted and a protein structure was solved. Of course under these conditions, crystals haven't grown out of the haze yet. Crystallizing the more conformationally inflexible tight binding DNA complex is more difficult than it logically should be.
Is there a term for this white haze or any information I can read about it?
What does it suggest to you?
Thanks!
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To me, this looks like amorphous precipitate of your protein and maybe a sort of "crust" or skin of mother liquor that covers the droplet during vapour diffusion. I'm not sure what you are looking for, but this specific condition does not contain any crystals, and the precipitate does not really look pre-cristalline.
How did you come up with this condition? Have you tried using commercial screens to determine a suitable crystallisation condition before optimising this specific buffer combination? You usually don't optimise until you actually have a hit condition.
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What happens when we culture cancer epithelial cells in TCPS?. Is the cell present as epithelial or differentiate into mesenchymal cells?.
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Aug 2, 2016 - Three-dimensional (3-D) cultures of cancer cells can potentially bridge the gap ... alternative to 2-D cell culture [3] as they can reproduce many aspects of the tumor ... to cells grown on 2-D tissue culture polystyrene plates (TCPS). ... If you allow us to do so, we also inform our social media, advertising and ...
by SP Lamichhane - ‎2016 - ‎Cited by 32 - ‎Related articles
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Hello fellow scientists,
I have some additional questions about the dissociation constant for a protein binding ligand.
My main questions is if the protein concentration is much greater than the KD and the ligand concentration is still in excess of the K_D how much protein is bound?
For a Protein binding a ligand, we have the relationship:
P + L ⇌ PL . This has forward and reverse rates of binding, and ...
KD = {[P] * [L]} / [PL],
The fraction of protein bound (FPL) will be:
FPL = [PL] / ([P] + [PL]) = {[L] / ([L] + KD)}
If the ligand concentration is some multiple integer n of the KD we get this cool relationship:
[L] = n * KD ; n = 1, 2, 3, ... etc.
FPL= (n * KD) / (n*KD + KD)
= n / (n + 1)
What they teach you in school is that if the ligand concentration is at the KD, n = 1 then half the protein is bound: 1 / (1 + 1) = 1 / 2. This relationship also tells you that if [L] = 9 * KD then 90% of the protein is bound.
One can plot this relationship as I have.
So I have some questions assuming a reasonable good ligand interaction with KD = 10 µM,
(A) If [L] = 9 * KD, and fixed protein concentration = KD = 10 µM, how much protein is bound?
I think that 90% of the protein is bound.
B) Say you are a structural biologist and you need more ligand and protein than the KD at 10 µM,
If [L] = 500 µM (that is 50KD) and initial protein concentration is 100 µM (that is 10KD), then how much protein is bound?
Given the relationship I derived, {50 / (50 + 1)} = 98% of the protein should be bound.
But the protein concentration is beyond the KD so can that even be correct?
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Here is the problem. In the math above, [L] is not the total ligand concentration. It is the free ligand concentration. If [P] is near or above the Kd, and [L] is not >> [P], then the free and total ligand concentrations will be significantly different, and the mathematical relationships above will give incorrect results.
In the so-called tight-binding domain, which is the situation just described, you should calculate [PL] using the tight-binding (Morrison) equation.
FPL = {(Kd + PT + LT) - square root[(Kd + PT + LT)2 - 4PTLT]}/(2PT)
where PT andLT are the total concentrations of P and L.
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Dear all,
I am wondering if someone uses a single point or double point method to determine the intrinsic viscosity of their proteins.
These methods are usually developed for polymers and does not fit for proteins.
Could you share with me your experimence on this.
Best,
Sébastien
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This question is addressed to biochemists and biophysicists, including those thinking about the conditions of living matter origination, thermodynamics of biochemical processes, physical phenomena underlying them, and chirality nature and origin.
We showed that the processes that leave to formation of precursors of nucleic acids, DNAs and RNAs and living issues, and the processes of formation of cells proceed on the basis of the internal energy of the molecules which participate in these processes.
This doesn’t mean that some simple elementary reactions can’t proceed cumulatively in such a way when a thermodynamically impossible reaction proceeds simultaneously with and at the expense of another reaction that has the excessive decrease in the free energy for its own proceeding.
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Dear Colleagues,
Apparently, a trigger is desirable for starting the discussion.
The discovery mentioned in this question means that the thermodynamic and chemical grounds of the theoretical biology should be revised. It is possible to turn a blind eye to this fact. But this will be made sooner or later.
The notion of the synthesizing of biological products through the preliminary synthesis of adenosine triphosphate for using its energy in subsequent syntheses of different bio-substances is commonly accepted. But what is the reaction that gives the energy necessary for ATP synthesis? Maybe it makes sense to include this reaction into the bio-cycle, if it exists and is identifiable? Not only thermodynamically impossible final biological products but also thermodynamically impossible intermediates cannot be synthesized. Protein, apparently, can be synthesized without ATP; this is shown in one of our papers listed below. What particular products entering into the bio-cycle cannot be synthesized without ATP? Maybe it makes sense to return to this question? If a set of source substances is thermodynamically capable of forming a product and the process of its formation is multistep, the synthesis of a final product should proceed through stepwise decreasing in the free energy. On the contrary, if synthesis of a product from a set of source substances is thermodynamically forbidden, no change in the sequence of reactions between these source substances is capable of synthesizing the product.
Enzymes, thousands of different types? What are the forces and circumstances that compel them to be synthesized? By what way are they capable of occurring in a necessary moment at a necessary point? What is about the diffusion laws? What makes them do the work that is attributed to them?
The first physicochemical explanation of the phenomena of DNA replication and mitosis was given in our works available in my ResearchGate pages by the addresses:
and in other our works on this problem.
Don’t you like our explanations? Why?
Please, propose another physicochemical explanation of these phenomena.
Without an adequate physicochemical description of a phenomenon, there is no science.
Prof. Hervé Seligmann, a scientist highly experienced in different biological problems, the author of many important publications, and the editor of the book “DNA Replication – Current Advances” (2011), wrote in the Preface to this book:
«But perhaps the most astonishing and challenging novelty in this book is the approach of DNA structure and chemical dynamics by focusing on its interactions with its natural solvent, the water molecules, and the changes in water concentrations through the cell’s life cycle (chapter by Ostrovskii and Kadyshevich). This most interesting hypothesis develops a concept that escapes conceptions established through the force of habit, which frequently result in dominant, yet unproven intuitive truths. This hypothesis will doubtlessly produce new deep insights into every level of DNA associated processes, and probably also general cell physiology, if given the deserved consideration and further developed. The approach in that chapter integrates processes associated with DNA and its structure with more basic physical properties at a lower scale of natural phenomena, namely the multimolecular dynamical structure of water.»
However, up to now, I don’t see bold spirits, which are ready to devote their time and energy to development of physicochemical understanding, including thermodynamic grounding, of driving forces of those processes which underlie the life development and dying. I ascribe this situation to the system of financial support of the mainstream hypotheses to the prejudice of principally new ideas, what was always a feather in science's cap.
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Wikipedia describes Physics, lit. 'knowledge of nature' , as the natural science that studies matter, its motion and behavior through space and time, and the related entities of energy and force
But isn’t this definition a redundancy? Any visible object is made of matter and its motion is a consequence of energy applied. We might as well say, study of stuff that happens. But then, what does study entail?
Fundamentally, ‘physics’ is a category word, and category words have inherent problems. How broad or inclusive is the category word, and is the ordinary use of the category word too restrictive?
Is biophysics a subcategory of biology? Is econophysics a subcategory of economics? If, for example, biophysics combines elements of physics and biology, does one predominate as categorization? If, as in biophysics and econophysics and astrophysics there are overlapping disciplines, does the category word ‘physics’ gives us insight about what physics studies or obscure what physics studies?
Is defining what physics does more a problem of semantics (ascribing meaning to a category word) than of science?
Might another way of looking at it be this? Physics generally involves the detection of patterns common to different patterns in phenomena, including those natural, emergent, and engineered; if possible detecting fundamental principles and laws that model them, and when possible using mathematical notation to describe those principles and laws; if possible devising and implementing experiments to test whether hypothesized or observed patterns provide evidence for or give clues to fundamental principles and laws.
Maybe physics more generally just involves problem solving and the collection of inferences about things that happen.
Your views?
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If you ask a fake physics :
  • Why is the sky blue? He says because it looks blue.
  • Why is the electron charge quantized? He says because Millikan's experiment has shown.
  • Why is there no ether? He says it was not shown in the Michelson's experiment.
  • Why is light a wave? Because Yang's test results are more consistent with light waves.
  • What is quantum mechanics? Like the great Feynman! He says he doesn't know, but he has accurate calculations and is compatible with the data, and that's enough.
The latter was not the answer of an ordinary physicist, but the answer of one of the greatest contemporary physicists! And this is a disaster for physics.
It is as if the role of physics has been reduced from a master to a servant.
Is reducing the role of physics from describing nature to a tool for exploitation a service to physics or a betrayal of it?
Technology is now far ahead of knowledge, and physics does not seem to be afraid of this humiliation, and it is still content with its instrumental role.
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Hi everyone,
I need to do MD simulation of wild type and ten variants at 50 ns. I am looking for a low-cost cloud service/ simulation environment. Would you please suggest me any?
Thanks in advance.
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you can check docker: https://www.docker.com/
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Hi,
I have been awarded the DBT-CTEP travel grant to attend the conference "64th Annual Meeting of the Biophysical Society" in San Diego which is going to be held between 15th to 19th February, 2020.
I would like to seek clarification regarding the travel dates. I have submitted the fare certificate as requested in the travel grant application which mentions that the travel duration is between 12th to 20th Feb, 2020 along with the fare details.
Now I am planning to visit few laboratories in US after the conference which will be helpful for my current research work. As I will be travelling by the same route (same fare) mentioned in the fare certificate, I would like to enquire if DBT will reimburse the travel fare if I change my duration of stay in US to 13th to 27th Feb, 2020. As this duration is different from the fare certificate, I would like to make sure that DBT doesn't reject my grant approval.
If anyone has information about this issue, please let me know.
Regards,
Sumanth
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Possible ly you may enquire from DBT Office
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So I have some pH = {4, 7, 10} standard buffer solutions from Fisher Scientific. But they must have went bad or I must have contaminated them because when I try to calibrate my pH meter to 4 using the pH 4 standard buffer solution, it calibrates the pH meter to 2 which obviously isn't right because it should be 4 so I can't trust the standard buffer solutions!!!
I need to do some experiments which require that I accurately calibrate the pH meter which means I can't titrate the standard buffers with HCl or NaOH. I have to know the exact quantities. Might seem a little over-the-top, but I want to be really certain about the math.
I intend to prepare my own calibration standards using exact quantities of chemicals. Total concentration should be 0.1 M, volume needs to be 100 mL. I think 0.1 M is a good choice because that should have low ionic strength and not much crazy debye-huckel variations in pH.
I am pretty certain that I know how to prepare a pH = 4 standard buffer solution using acetic acid and sodium acetate. But I am not quite sure how to prepare a pH 7 standard.
First of all, is this exactly how I should prepare a 0.1 M acetate buffer in 100 mL?
According to pubchem the pKa of acetic acid is 4.76. I have a chem textbook which tells me that the pKa is 4.74. Not much difference so I will assume pubchem is right.
HA + H2O --> A- + H3O+
Acetic Acid + H2O --> Acetate + H3O+
Henderson-Hasselbalch Equation: pH = pKa + log([A-] / [HA])
pH = pKa + log([Acetate] / [Acetic Acid])
= 4.76 + log([Acetate] / [Acetic Acid])
For the total concentration:
[Acetic Acid] + [Acetate] = 0.1 M
The simultaneous solution to these equations is about:
[Acetic Acid] = 0.0852 M
[Acetate] = 0.0148 M
pH = 4.76 + log(0.0148 / 0.0852) = 3.9998 etc ... Pretty much exactly 4!
I have glacial acetic acid and sodium acetate.
Glacial Acetic acid is a pure liquid so its concentration is its density / (Formula Weight)
[Glacial Acetic Acid] = [(1.05 g/mL) / (60.05 g/mole)] * (1000 mL / 1 L) = 17.5 M
Basic Dilution Equation:
Initial Volume = (0.0852 M / 17.5 M) * 100 mL = 0.487 mL glacial acetic acid in 100 mL total volume.
Sodium Acetate --> Na+ (aq) + Acetate- (aq)
grams sodium acetate = (0.0148 moles sodium acetate / L) * (82.03 grams sodium acetate / 1 mole sodium acetate) * (1L / 1000 mL) * 100 mL = 0.121 grams sodium acetate
So I can exactly prepare a pH = 4 is 0.1 M acetate solution by adding 487 microliters of glacial acetic acid & 0.121 grams sodium acetate to 100 mL volumetric flask, and bring the volume up to 100 mL with ultrapure deionized water? Swirl to mix.
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I am confused about how to prepare an exactly pH = 7 phosphate buffer solution.
I know the step wise dissociation of Phosphoric Acid. But the problem is that I am not certain about the second dissociation constant.
H3PO4 --> H2PO4- (aq) + H+ (aq)
H2PO4- (aq) --> HPO42- (aq) + H+ (aq)
If I know the second dissociation constant of phosphoric acid I could figure out exactly how to make a pH = 7 standard 0.1 M phosphate buffer solution using phosphate salts.
My undergrad general chem text book is telling me the second Ka is 6.2 * 10^(-8) or the pKa is 7.208 and pubchem is telling me the second the second pKa is 7.09.
Thus I could use the Henderson-Hasselbalch Equation again with the total concentration to figure out the exact quantities. But I am not certain about what the second pKa of phosphoric acid is! Are undergrad chem textbooks just wrong? That's too much of a margin of error.
pH = pKa + log[(K2HPO4) / (KH2PO4)]
Also I was wondering if a 0.1 M solution of NaCl would have a pH of EXACTLY 7? Would it be more or less accurate than making the phosphate buffer?
NaCl is formed by the titration of a strong acid HCl with a strong base NaOH. Therefore the equivalence point of the titration of a strong acid and strong base should yield a pH 7 of neutrality forming NaCl.
So if I dissolved NaCl in water to yield a 0.1 M concentration would the pH be just as good as making that phosphate buffer solution?
I also need to figure out how to make a pH = 10 standard buffer solution.
Any advice? Can you make my life easier?
Thanks so much!
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There must be some problem with the pH meter then.. U should show it to a technician so that it can be fixed.
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I do like Cell, Nature, & Science. I find the discoveries to be amazing. But the experiments can be difficult to understand and difficult to replicate without the state of the art scientific instruments. So I have been frequently reading the volumes of Methods in Molecular Biology and Methods in Enzymology. The reason is because these journals give reproducible experiments with simple explanations.
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Few of us wanted to create a discord server for Biophysics. What we intend is to begin a commonplace for discussions/numerical experiments. Also possibly document the results in the form of blogs or other media.
I believe that there are many biophysics/computational biophysics/Molecular Dynamics enthusiasts here. Here is the server link: https://discord.gg/qRQRq2k
Come and join us. Let us learn together.
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Dear Devanand,
Can you explain more what is the purpose of this post and what the discord means?
Bog
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"An orgone accumulator (ORAC) was a box developed by Wilhelm Reich to trap and then radiate his magical orgone energy into a concentrated area. Reich constructed the box so that it had organic material lining the outside of its walls and metallic material lining the insides of its walls. To Reich orgone energy would be drawn into the organic material on the outside of the box but then be radiated into the box by the metallic inner lining. Over time this orgone energy would "accumulate" in the interior of the box. "
Are there current publications about the Orgone accumulator? If yes, can you share this section?
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TO start with Reich was charged with contempt in 1956 for having violated an injunction to transport his orgone energy synthesizer across state lines in the USA. He got a two-year prison sentence , and six tons of his publications were burned by order of a federal court. He died in a US Federal prison of heart failure at age 60. by any standard, he was not psychologically healthy. However, the journals that publish this material whilst largely non-indexed or ranked, do include:
DeMeo, J. (2009). Experimental confirmation of the Reich orgone accumulator thermal anomaly. Subtle Energies & Energy Medicine Journal Archives, 20(3).
Correa, P. N., & Correa, A. N. (2001). Comparative study of the variation in the spontaneous discharge rate of atmospheric electroscopes and electroscopes placed within orgone accumulators'. Experimental Aetherometry, 1(6), 1-81.
Klee, G. D. (2005). THE RESURRECTION OF WILHELM REICH AND ORGONE THERAPY. Scientific Review of Mental Health Practice, 4(1).
Rosch, P. J. (2009). Bioelectromagnetic and subtle energy medicine. Annals of the New York Academy of Sciences, 1172(1), 297.
Mazzocchi, A., & Maglione, R. (2010). A preliminary study of the Reich orgone accumulator effects on human physiology. Subtle Energies & Energy Medicine, 21(2), 41-50.
Morgavi, G., Morando, M., Biorci, G., & Caviglia, D. D. (2005). Growing up: emerging complexity in living being. Cybernetics and Systems: An International Journal, 36(4), 379-395.
Martin, W. J. (2015). Interacting light paths attract KELEA (kinetic energy limiting electrostatic attraction) and can lead to the activation of water. Open Journal of Biophysics, 5(04), 115.
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cancer (oncology) is field of biophysics
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Yes, there is a relationship. I cannot explain it very well, though.
Cancer cells respond to pressure on the membrane by using it as a signal to grow all the more. Normal cells interpret the signal as to stop growing. Other than cancer cells there are other cells which deal with a lot of stress. Heart cells experience a lot of pressure as blood rushes in and out of them.
The pathways that say affect cancer through the various types of pressure waves on the membrane are hard to describe because integral membrane proteins in the membrane send the signal through a kinase cascade resulting in signaling transcriptional co-activators being imported into nucleus which bind to transcription factors and result in the expression of genes that reprogram the cells.
Of course this is all so vague. But what is interesting is that the transcriptional co-activator yes-associated protein's (YAP) level of nuclear expression governs the level of cellular stretching. High pressure also activates YAP resulting in pretty much the same phenotype. The mechanism how this happens is unknown. There is a see of proteins involved in the Hippo Pathway. It is also possible that the pressure waves are transmitted through the cell and flatten the nucleus. This has been studied just a bit, but as you can imagine it is difficult to study the architecture of the nucleus inside a living cell with an intact external membrane.
This article doesn't mention YAP. It is a big puzzle.
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I am trying to plan an pulldown assay to find some prey protein for my bait protein.
I am interested in using the flag tag because it is small and doesn't disturb structure and doesn't require additional steps to tag your protein like would be necessary with biotinylation.
Plus apparently, the bait-prey complex can be eluted with Flag peptide.
So that is very interesting to me.
However, I haven't been able to figure out how strong Flag-Tag binding is to Anti-flag antibody?
Also, does anyone have a simple protocol for Flag-tag pulldown?
Flag Tag = DYKDDDDK
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In my hands, Flag antibody is OK but not the best. I find HA is cleaner. One thing to consider in IPs with Flag is the amount of DTT in the lysis buffer. DTT weakens the interactions. I now don't add DTT in my lysis buffer if doing IPs with Flags.
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Lysozyme is consisted of 4 disulfide bridges while the number of disulfide bridges for BSA is 17. Nevertheless, lysozyme is known to be a more rigid protein. What are the other factors affecting the rigidity of proteins?
Does amount of a specific secondary structure like alpha-helix or beta-sheet necessarily determine the rigidity of a protein?
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Lysozyme is much smaller (Structure 1HEN 129 amino acids) than serum albumin (structure 1AO6 585 amino acids). Lysozyme can be considered a single domain, while serum albumin consists of several subdomains (see Fig.1 in ).Flexibility is part of the function of serum albumin, it serves as carrier for many different hydrophobic compounds, that do not utilize one define binding site, but intercalate in many different sites into the hydrophobic core of the molecule. While secondary structure (hydrogen bonds) and disulfide bonds certainly have an effect on flexibility, core packing also has a major effect. If you look at the localization of the disulfide bonds within albumin, you will see that they are quite local in the individual subdomains, keeping the helices of the subdomain together, but allowing the different subdomains to move relatively to each other.
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I am doing research on indentation simulations. And I don't want to use all atom MD simulation. Is there any coarse-grained indentation simulation example?
Thanks!  
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Yes coarse grain with martini is good option. But you must know why your opting same. If your system is big or contains bilayer then you can use INSANE to construct the bilayer too.
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Please explain concisely, just like Nucleus can be cellular nucleus or atomic nucleus from perspective of biophysics
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Cold Atmospheric Plasma is an ionized gas that has recently been extensively studied by researchers as a possible therapy in dentistry and oncology. Several different gases can be used to produce  Cold  Atmospheric Plasma such as Helium, Argon, Nitrogen, Heliox, and air.
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Although biochemistry text books define pKa values for each residue, the actual pKa of a residue depends on several factors: location in the protein, presence of ions etc.
So, given a protein structure and environment, is there a way to predict the pKa of a given amino acid?
Thanks
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Yes, the pKa range for a given amino acid in proteins can be found in the literature. But for predicting the pKa for a particular residue in a particular protein, a computational method is needed.
PROPKA is a well-established tool for this purpose.
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I have Amyloid beta 1-40 monomer using which i need to perform biophysical characterisations.
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Thank you Saranya Sivaraj and Mahesh Chandak for your answer
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I am having trouble calculating free energy of folding, enthalpy and heat capacity of my peptide samples. I performed a thermal unfolding of the peptides, and obtained the ellipticity in mdeg.
Next, I converted the mdeg to mean residue ellipticity using the equation:
m.r.e. = mdeg / [path length, mm * Molar conc. * Number of residues].
Is this correct?
With Origin software, I used a Boltzmann's fit to determine the Tm of the peptides. But how can I calculate the enthalpy, heat capacity and free energy of unfolding assuming a 2-state model?
Which software permits me to do this? And how can I actually use equations to compute the mathematical simulations?
All suggestions would be appreciated.
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Using the Boltzmann function is not a good way to analyze protein unfolding assays. This curve has an appropriate shape, but its parameters (inflection point location, difference between asymptotes, asymptotes) may not have any relation with the thermodynamic parameters you would get from the correct analysis (Tm, unfolding enthalpy, unfolding heat capacity) from which you can compute the stability profile (Gibbs energy of unfolding as a function of temperature). Even the temperature for maximal slope (center of the transition) may not correspond with the actual unfolding transition temperature (Tm). Therefore, the Boltzmann fit is an example of a curve that may go through the experimental points, but gives no information at all about the stability of the protein.
You should analyze the unfolding experiment using the two-state or multiple-state (non-two-state) unfolding model. You can use any data analysis software (Origin, Excel, KaleidaGraph, SigmaPlot, DataFit, LabPlot...), because all these packages will allow you defining user-defined fitting functions.
But, first, you should read articles, book chapters, and protocols where the two-state or the non-two-state models are described or employed.
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I am using AMBER for MD. However I am not able to figure out how to select atoms or residues to apply a harmonic restraint.
I have crystal structure from PDB and and I want run MD simulations on it. At the active site there is a Zinc ion and I want it remain there along with surrounding residues, water molecule and ligand so that it stays there in similar conformation during MD run. I don't want it to fly away. Subsequently I want to run QM simulations on the same systems.
Is there any way to restrain the atoms and residues within a particular distance from specific atom e.g. zinc?
my pdb is 2v5w
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Interesting question! But im not sure, if you restrain the Zn ion in the pocket, what suppose should be the consequences. They wouldnt take part in the dynamics. As other suggested you can go for bonded MD in If you using AMBER.
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I am currently analyzing the impact of a policy instrument on deforestation rates. The unit of analysis is a landscape (geographically selected based on biophysical conditions). I have eight landscapes (8 cases/observations), four of them have the policy instrument (participants inside the landscape have voluntarily decided to join the forest conservation program) and four don´t. Each landscape has diverse information (continuous, Likert scale, percentages, etc.); for example, there is income information from 800 (100 in each landscape) households but also governance indicators (Likert scale) from 8 (one in each landscape) communities. Due to the variety of information at different levels across the landscapes I decided to use average values for the each variable in all landscapes. I face now the following problem: I have 8 observations but more than 50 independent variables.
Questions:
Is there a statistical technique that accounts for too few cases and too many variables?
Except for backward elimination, how could I select the appropriate variables for analysis? LASSO?
Also, considering the number of observations, is there a threshold for statistical values (R2, P-value, t-statistics, etc.) that I must meet to have significant (publishable!) results from the regression?
Thanks a lot!
Fernando
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With so many variables, it will overfit. According to my limited knowledge, you can perform Principal Component Analysis and decrease the number of variables.
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I am currently looking for academic professional that can accept my studentship for PhD program in any of the following research area:
1. Antimalarial or anticancer novel compound synthesis and biochemical evaluation.
2. Structural biology of uncharacterized enzyme target for malarial or cancer therapy.
3. And other related research topics.
These studies will involve the use of bioinformatics/computational and biochemical/biophysical techniques.
Country of interest: Brazil, USA, Germany and other available countries.
Kindly let me know your willingness
Kind regards.
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Do you have your own funds?
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We are working on the fibrillation of lysozyme. Given the acidic condition (pH 2) of the protein solution (glycine buffer) for fibrillation, what should be buffer and its pH for ThT stock solution?
Should the buffers of protein and ThT solutions be identical?
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I have never worked at such low pH with ThT. The best way would be to put ThT in water or in the same pH of your buffer but the efficiency of the absorption and emission will change with respect to physiological pH. I would suggest you to give a look at this paper (DOI 10.1007/s00249-015-1019-8 ) in which ThT was tested in a wide pH range.
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Hello,
I am working on a biophysics core facility where we have developed a service to check among other things the molecular mass of the protein of interest, the presence of contaminants and degradation (MALDI-TOF).
I used to work with sDHB as a matrix for protein between 10 and 20 kDa and with sinapinic acid for proteins >20 kDa.
These two matrices are dissolved in 50% ACN, 0.1% TFA in water and protein buffer can be so different according to the protein.
I have no real difficulty to get nice spectra on our UltraFleXtreme mass spectrometer. However my main difficulty is to get rid of of adduct peaks such as TFA adduct. You can find attached an example of spectrum that I used to get.
What is your experience to improve that?
Best,
Sébastien
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Dear Sébastien,
Despite the protein you analyzed is produced by bacteria, it looks very like that you have an glycoprotein. MW of the common monosaccharide residue, N-acetylglucosamine (or N-acetylhexosamine) is about 203 Da that has a good correlation with your data. Try to treat you sample with exoglycosidase that cuts this residues (for example, β-N-Acetylglucosaminidase) and repeat the analysis.
But in a more classical way, firstly you need to do is to measure MW with a better mass accuracy and resolution (ESI-TOF or orbi) and secondly perform a bottom-up approach with several proteases to reliable identify this modification.
Best wishes,
Maksim Degterev
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Considering that mean field theory approaches have been used for neuronal dynamics, and that renormalization group theory has been used in other networks to describe their properties, I wanted to know whether it is useful or interesting to describe the behavior of a neuronal system based on its critical exponents. Thank you in advance.
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Critical exponents describe properties that don't depend on whether the network describes brain function or some other system.
If the network does show scale free behavior, then critical exponents can be defined and they characterize the behavior of certain quantities.
That's why it's wrong to ask whether it's useful or interesting to describe the behavior of a neuronal system based on its critical properties-but whether it does have such properties at all.
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Dear All,
As we all know that lower Kd (micro-molar/nano-molar range) are considered as good interaction. But how one can appreciate the relative concept of this strength when I am measuring a single pair of protein-protein interaction. Please provide your valuable remarks on this. Thank you in advance.
Prem
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The strength of a given interaction can be judged through the association constant K or the dissociation constant Kd. Very roughly, and taking 1 M as the reference standard state concentration:
- Low affinity: Kd larger than 10-4 (> 100 microM)
- Moderate affinity: Kd between 10-4 and 10-7 (100 microM - 100 nM)
- Low affinity: Kd smaller than 10-7 (< 100 microM)
However, people working on in different fields may have different considerations. Thus, a Kd of 10-6 (1 microM) can be considered as high affinity in metabolism regulation, while it can be considered a low affinity in antibody design.
And this is related to another way to judge the strength of an interaction which takes into account the potential concentrations of the interacting molecules. Then, a Kd of 10-6 (1 microM) has different physiological consequences if the interacting molecules are present at mM (complex predominates), microM (complex and free molecules at comparable molar fractions) or nM (free molecules predominate) concentrations.
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I just wants to measure the protein sample using THz Spectroscopy. Can anybody suggest me what concentration is appropriate??
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What is the most important problem in the computational physics, especially in biophysical domain.
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Signaling network procedure from brain to nerves, associated
with every organs . Biophysics of Brain / Neural Network Modelling .
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Hello!
For instance, in MD simulation of the the interaction between a peptide and a metal ion (like Zinc ion, Al ion, Mg ion etc.), does it possible to define the peptide as ligand and the metal ion as receptor?
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The binding free energy is the free energy difference between the complex and the two isolated components. This is independent of which of the two components you call receptor and which you call ligand.
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Dear Colleagues,
I am relatively a newcomer to the amazing fields of photophysics and photochemistry.
From the available scientific literature, we may read that induced chlorophyll a fluorescence is mainly emitted by chlorophyll a molecules, located in Photosystem II (PSII), upon illumination onset. It has been reported about 300-500 chlorophyll a molecules in a single Photosystem II.
PSI fluorescence is constant and much lower than fluorescence from PSII. Its contribution to emitted plant fluorescence is considered negligible.
Some authors speak about P680 (a pigment named P680, located in Photosystem II), the reaction center RC or the special pair or the special chlorophyll dimers pigments PD1 or PD2, as the only source of fluorescence. I have a bit of confusion because it is not clear what chemical species is emitting the fluorescence that we can sense with our portable fluorometers.
1) If the Special Pair or RC is closed (it has been chemically switched to its reduced state), during the time that the Special pair is in that state, is the full bunch of chlorophyll a molecules in PSII going to dissipate their excitonic energy as fluorescence?
2) Why fluorescence emitted from PSI is not variable but constant? Does it has this fact something to do with the ratio [Chl a] to [Chl b] ???
Thank you so much in advance for your precious and kind help!
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Howdy Stancho,
First Chlrorphyll fluorescence is not a photophysical or photochemical phenomenon, but a photobiological one.
Chlorophyll fluorescence is light re-emitted by chlorophyll molecules (yes
chlorophyll!) during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in higher plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis (photochemical energy conversion), as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectra of applications.
The Kautsky effect
Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem II (PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect. This variable rise in chlorophyll fluorescence rise is due to photosystem II. Fluorescence from photosystem I is not variable, but constant.
The increase in fluorescence is due to PSII reaction centers being in a "closed" or chemically reduced state. Reaction centers are "closed" when unable to accept further electrons. This occurs when electron acceptors downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more "photochemical quenching" in which electrons are transported away from PSII due to enzymes involved in carbon fixation; and 2. more "non-photochemical quenching" in which more energy is converted to heat.
PSII yield as a measure of photosynthesis
Chlorophyll fluorescence appears to be a measure of photosynthesis, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport by multiplying with light intensity. However, researchers generally mean carbon fixation when they refer to photosynthesis. Electron transport and CO2 fixation correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.
Relating electron transport to carbon fixation
A powerful research technique is to simultaneously measure chlorophyll fluorescence and gas exchange to obtain a full picture of the response of plants to their environment. One technique is to simultaneously measure CO2 fixation and PSII photochemistry at different light intensities, in non-photorespiratory conditions. A plot of CO2 fixation and PSII photochemistry indicates the electron requirement per molecule CO2 fixed. From this estimation, the extent of photorespiration may be estimated. This has been used to explore the significance of photorespiration as a photoprotective mechanism during drought.
Fluorescence analysis can also be applied to understanding the effects of low and high temperatures.
  • Sobrado (2008) investigated gas exchange and chlorophyll a fluorescence responses to high intensity light, of pioneer species and forest species. Midday leaf gas exchange was measured using a photosynthesis system, which measured net photosynthetic rate, gs, and intercellular CO2 concentration. His results show that despite pioneer species and forest species occupying different habitats, both showed similar vulnerability to midday photoinhibition in sun-exposed leaves.
Measuring stress and stress tolerance
Chlorophyll fluorescence can measure most types of plant stress. Chlorophyll fluorescence can be used as a proxy of plant stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a plant to metabolise normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.
  • Favaretto et al. (2010) investigated adaptation to a strong light environment in pioneer and late successional species, grown under 100% and 10% light. Numerous parameters, including chlorophyll a fluorescence, were measured. Overall, their results show that pioneer species perform better under high-sun light than late- successional species, suggesting that pioneer plants have more potential tolerance to photo-oxidative damage.
  • Neocleous and Vasilakakis (2009) investigated the response of raspberry to Boron and salt stress. Leaf chlorophyll fluorescence was not significantly affected by NaCl concentration when Boron concentration was low. When Boron was increased, leaf chlorophyll fluorescence was reduced under saline conditions. They be concluded that the combined effect of Boron and NaCl on raspberries induces a toxic effect in photochemical parameters.
  • Lu and Zhang (1999) studied heat stress in wheat plants and found that temperature stability in Photosystem II of water-stressed leaves correlates positively and well, to the resistance in metabolism during photosynthesis.
Nitrogen Balance Index
A portable multiparametric fluorometer using the ratio between chlorophyll and flavonols can be applied to detect nitrogen deficiency in plants Because of the link between chlorophyll content and nitrogen content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency in plants, by several methods.
Based on several years of research and experimentation, polyphenols can be assigned as indicators of the nitrogen status of a plant. For instance, when a plant is under optimal conditions, it favours its primary metabolism and synthesises the proteins (nitrogen molecules) containing chlorophyll, and few flavonols (carbon-based secondary compounds). On the other hand, in case of lack of nitrogen, we will observe an increased production of flavonols by the plant.
The NBI (Nitrogen Balance Index), allows the assessment of nitrogen conditions of a plant by calculating the ratio between Chlorophyll and Flavonols (related to Nitrogen/Carbon allocation) .
Hence Stancho,
I hope this short summary elucidates soma aspects of the photobiology of plant chlorophyll fluorescence. A lot more information is available. For example models to simulate plant fluorescence. An interesting one can be found at:
Success with your studies,
Frank
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If we have an unknown cell, for basic studies on its properties, we need to detect the combination of channels on the cell.except voltage clamp method,how we can do that?
for example If we have a new and unknown disease,we need to detect the combination of channels on the cell for Eliminating the disease agent.
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Please see the reference that would be useful for this issue.
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I am wondering if there are any web-based programs, like ExPASy that can "predict" a protein's sequence based on structural input. For example, ExPASy SWISS-MODEL can predict protein structure based on sequence input, but does the vice versa exist, either on ExPASy or elsewhere?
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Depending on what your need is - if you are working with a structure that has been deposited in the PDB, you may want to use the sequence associated with that file in the PDB, which (usually) is the sequence of the construct used - including residues that are not resolved in the structure because of disorder. This sequence is also to be found in the SEQRES records in the header of the PDB file. If you need exactly the sequence represented by the coordinates, the servers listed by
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After reading related papers, I'm honestly a bit lost about how to use the PROSAIL model (What is it really for? How do I get the biophysical parameters? Can I use it only with reflectance values? and so on).
I was wondering if anyone has a guide of some sort or reference material that's easier to understand, or if there is anything essential I should read before going deep in the topic.
Best Regards and thanks in advance!
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