Localization - Science topic

Hi, the answer provided by Prof. Janak Trivedi is pretty comprehensive, agree with that. The ideal approach would be to capture equal number of slices for each stack, but I guess some samples have the signal spread over a greater depth (axially) so you don't want to miss out that signal. Also, you mentioned you make "a maximum intensity profile of Z- stack". So I suggest you average out and also make a montage of your stacks (ImageJ options) and then compare the intensity profiles. Additionally. check out this article:

Hope it helps.

Sonashree Ss There are numerous methods for expressing a protein of interest in cells for live imaging without interfering with endogenous protein localization:

1. Utilizing a particular promoter: You may verify that the production of your protein of interest is confined to the desired region by using a promoter that is only active in a certain cell type or subcellular location.

2. Utilizing a conditional expression system: You may regulate the expression of your protein of interest by using a system such as the tetracycline-inducible system, which allows you to switch it on and off at certain moments.

3. Using a degron system: By fusing a protein of interest with a degron, you may activate its breakdown at specified moments, guaranteeing that your protein of interest is expressed.

4. Using a fluorescent protein: You can fuse your protein of interest with a fluorescent protein to view it while leaving endogenous proteins alone.

5. Using a particular tag or epitope: You can attach a specific tag or epitope to your protein of interest, allowing you to detect it precisely with an antibody while avoiding cross-reactivity with endogenous proteins.

It is worth noting that certain approaches may be more appropriate than others depending on the specific protein, cell type, and imaging system used. To determine the ideal technique for your experiment, it's always a good idea to research the literature and test several strategies.

Hi David Pla-Martín,

Thank you so much for your answer!!

I'll try reducing the concentration of PK by serial dilutions and optimize it!

Do you have access to cadavers? If so, that would help. Regardless, recruit a physiotherapist/physical therapist to help you with locating the muscles. Electrode pair spacing on a given muscle should be no more than the thickness of the muscle.

Dear Ahmed MD Foysal ,

Minimally Invasive Surgery (MIS) is a surgical intervention used to examine the organs inside the abdomen and has been widely used due to its effectiveness over open surgery. Due to the hardware improvements such as high definition cameras, this procedure has significantly improved and new software methods have demonstrated potential for computer-assisted procedures.

Regards,

Shafagat

Dear Huazhong Wang

One plausible reason could be that most of the GFP derivatives form weak dimers under physiological conditions possessing low-affinity membrane-binding ability, particularly to phosphoinositide-rich membranes. On the other hand, mCherry is a monomeric FP, do not form dimers and do not interact with membrane.

Teresa Tommasini Have a look

Gaurav Kumar PSORT predicts protein localization locations using amino acid sequence information and guidelines established from experimental findings. Gram-negative bacteria, for example, have four localization sites: the cytoplasm, the inner membrane, the periplasm, and the outer membrane.

For example, when studying protein subcellular localization, two methodologies are typically used: fluorophore tagging of the protein (e.g., Green Fluorescent Protein) or a proteomic approach that identifies all the proteins inside a certain cellular compartment (e.g. Mass Spectrometry).

Xiao Yi NLSs are known to be involved in the active transport of foreign proteins and probes into the nucleus. However, the stimulation of plasmid DNA nuclear localization has yet to be fully validated.

Dear Keith,

I think AI helps path planning navigation system easier and powers self driving vehicle frameworks such as automatic detecting peoples and traffics.

Ashish

I don't think you can compute a band decomposed ELF with VASP, as in, I don't think IBAND and EINT are taken into account during the ELF evaluation.

If you want to be sure, you could take a look at the ELF module source code and grep for the key words IBAND and EINT.

Thank you all.

Dear Conner Murphy ,

first of all I would like to recomment the CellProfiler forum for specific problems. My experience there were great - a kind and responsive communtiy.

To come now to your problem. I would recomment to use a non specific stain for your cells. Drag5 might work to stain both cell body and nuclei. Otherwise you could use a CellTracker and Hoechst33342/DAPI to provide some spatial information of the cells. Than you should use identify primary objects to find your cells. (You should get now information for your staining per object (i.e. cell) Than I would use the DNA-staining to create a secondary object. These objects/masks can now be used to define the perinuclear space (by expanding and substration of the original mask).

Maybe you can post some sample images?

kind regards

Soenke

The following research article is related to this thread:

Best Regards.

Thank you for your suggestion Pablo Mtz !

Localization means discovering a region in the phase space, where the attractor is located. Usually, such a region lies in the basin of attraction. So, any point from the region tends to the attractor that allows to numerically compute it. Since for hidden attractors, their basins of attraction do not intersect with unstable manifolds of equilibria, one should develop an intuition for discovering such attractors. There are many developed approaches.

I recommend the following two surveys.

Monji Zaidi Anchor-based or anchor-free localization techniques are categorized as centralized or distributed, GPS-based or GPS-free, fine-grained or coarse-grained, stationary or mobile sensor nodes, and range-based or range-free. All of these strategies will be discussed briefly.

High bandwidth demand, high energy consumption, quality of service (QoS) provisioning, data processing, and compression methods, and cross-layer design are all challenges in such WSN. physical surroundings Mobile nodes, like static nodes, can perceive, compute, and communicate.

Zhu Han To influence their hosts, secreted effectors work in the apoplast or inside the cytoplasm of plant cells. Effector proteins can perform a variety of functions, but they often aid the pathogen in invading host tissue, suppressing the host's immune system, or otherwise assisting the pathogen in survival. Effector proteins are often required for pathogenicity.

Austin Morrissey A gene reporter experiment, which introduces an exogenous NF-B consensus promoter sequence connected to a measurable reporter gene' such as luciferase, is a typical approach for measuring NF-B activity at the transcriptional activation level.

A gene reporter experiment, which introduces an exogenous NF-B consensus promoter sequence connected to a measurable reporter gene' such as luciferase, is a typical approach for measuring NF-B activity at the transcriptional activation level.

NF-B targets genes involved in the onset and progression of inflammation. NF-B is a transcription factor that may be activated. After activation, it can control inflammation by activating the transcription of several genes.

Simpler and easy methods are using bright field microscope and flow cytotoxicity. u can check out these papers:

1. microscope:

2. flow cyto:

interesting discussion. I had not looked at vest rehab in this way. Now i may re review our patient data and see , if there is any such trends... atleast in new cases i can keep this in mind and see if there is any such issues.

Its also interesting to know that Ignacio Novoa Cornejo uses vHIT data , for rehab.

Do u use gain values to know is compensation has happned or not?...

Dear @Qamar Ul Islam, thanks for your response, however it is not the correct. Since the video explained the polar spiral or Archimedes spiral.

Thank you all for your valuable insights. I think I should have mentioned above that I have not got any high ELF value in the core of Pt atom for the antiferromagnetic arrangement of the system (done with same pseudopotential). The above-mentioned case was for ferromagnetic arrangement. Here I am attaching the AFM elf 2D pattern.

Yusuf Chong Yu Lok Make use of a vector and insert your gene in front of the GFP gene. Your gene's stop codon should be eliminated, and your gene and the GFP gene should be in the frame.

Dear Muhammad Usman,

Nanolithography (NL) is a growing field of techniques within nanotechnology dealing with the engineering (patterning e.g. etching, depositing, writing, printing etc) of nanometer-scale structures on various materials.

The modern term reflects on a design of structures built in range of 10−9 to 10−6 meters, i.e. nanometer scale. Essentially, the field is a derivative of lithography, only covering very small structures. All NL methods can be categorized into four groups: photo lithography, scanning lithography, soft lithography and other miscellaneous techniques.

For more information about this subject i suggest you to see links on topic.

Best regards

Open the cube file using vesta and you will see iso-surface, and in utilities go through 2D data display and click on the slice there you will find hkl plane select it along with the distance from the origin and apply. You will see the 2D slice in any plane you want and from any distance from origin.

As an affordable and accurate solution I would recommend the ”Underwater GPS“, that we are using with a BlueRov2: https://shorturl.at/floIJ

GetSensorsData suite

thank you for your suggestion,but the precesion is 98.8% and the recall is 97%,they are both high.@Qamar Ul Islam

Also see the following link: https://svi.nl/ColocalizationBasics

That is a good question.

Dear Oliver Wartmann

These articles might help, have a look:

1.

2. https://www.nature.com/articles/s41598-021-82798-w.pdf?origin=ppub

3.

Kind Regards

Dear Huachao Sun

These links might be useful, have a look:

1. https://jwcn-eurasipjournals.springeropen.com/articles/10.1186/s13638-018-1165-7

2.

3. https://www.mathworks.com/help/nav/ug/monte-carlo-localization-algorithm.html

Kind Regards

Dear Cristiane Matsuura

I suggest you go through these articles first:

1. https://www.microscopemaster.com/histochemistry.html

2. https://www.sciencedirect.com/topics/immunology-and-microbiology/silver-impregnation

3. https://pubmed.ncbi.nlm.nih.gov/29701111/

4.

5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048572/

6.

7. https://www.nature.com/articles/s41467-018-07676-y

8. https://iopscience.iop.org/article/10.1088/2053-1591/aa6922

Kind Regards

Dear Ayman A. A.

I recommend the following articles, have a look:

1. https://www.sciencedirect.com/science/article/pii/S0014482714000263

2.

3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732143/

4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556086/

5. https://www.frontiersin.org/articles/10.3389/fmolb.2020.00208/full

Kind Regards

You may want to start with this databases:

mpstruc is a curated database of membrane proteins of known 3D structure. To be included in the database, a structure must be available in the RSCB Protein Data Bank (PDB) and have been published in a peer reviewed journal. The database is manually curated based upon on-going literature surveys. Because of the labor involved, new structures are not posted until they are released by the PDB and the complete reference, including pagination for print journals, is available in PubMed. Our goal is to make mpstruc as accurate and complete as possible. If you find errors or omissions, please send a message to Stephen White, Gracie Han (gyewon.han@usc.edu), or Craig Snider. Stephen White, Gracie Han, or Craig Snider (Sorry, you seem to have javascript disabled). mpstruc emphasizes structures determined by diffraction and cryo-EM methods. NMR structures are also included whenever identified. A comprehensive list of NMR-determined structures has been established by Dror Warschawski and is available from the Atoine Loquet lab.

Membranome database provides structural and functional data about single-spanning (bitopic) transmembrane proteins of six organisms (human, thale cress flowering plant, slim mold, baker's yeast, gut eubacteria, thermophilic methanogenic archaeabacteria) representing all kingdoms of life.

It includes evolutionary classification, topology and intracellular localization of bitopic proteins, protein-protein interactions and known protein complexes, links to three-dimensional structures and metabolic pathways, and computationally generated models of transmembrane domains optimized in the lipid bilayer.

konnect2prot and cellmap are two excellent sites for subcellular localization identification.

Please refer to the following papers:

I recommend reading this paper to answer your question:

Interesting

Dear Neeraj Wasnik

These conversations might be helpful, have a look:

1. https://www.researchgate.net/post/What_is_the_best_percentage_protein_gel_for_a_16_KDa_protein_sample_using_Western_blot

2. https://www.researchgate.net/post/How_to_detect_small_molecular_weight_proteins_10_11_17_20_KDa_by_western_blot

Kind Regards

Dear C. B. , the simple and general answer to your first question should be clearly negative, the cellular uptake and cellular location of NPs are different experiments. However, they could be the same experiment if all the uptook NP´s are localized at the same organelle or cell region.

With respect to your second question, if you have two different drugs encapsulated in a polymeric delivery system to facilitate the uptake by the cell, they could be detected by confocal microscopy if these two drugs show distinctive fluorescence signals. It means that these two drugs must show auto-fluorescence in the conditions of excitation used and they must show different "colours" to be distinguished. If one or both drugs do not show fluorescence under the working conditions or they show the same or very close colours, then you must use some dye to label each drug. If your delivery system is intended for reaching some cell´s organelle and just there delivering its cargo, may be it could be possible to just follow the whole system (drug A + drug B + polymeric system) and labelling it with the addition of some fluorescent dye, well inside or attached to the polymeric shell. In this way you could see if the whole system reaches the cell and then the target organelle. To use this methodology you need to be sure that both drugs reach the target and that the polymeric shell keeps its cargo till the destination, in other case you would be following an empty- fluorescent- shell.

Hope this helps. Good luck with your research work.

Hello Varun Garg,

Equation (10) is a result of the non-additive noise formulation for the EKF:

The usual simplification is to add noise to the state after the dynamic/prediction step. Often, we have a better knowledge where noise in the system is present (in the paper example the noise on the velocity measurement ist known). In such cases, you can use the non-additive noise formulation.

If you want to prove/check covariance equations in general you can refer to covariance computation rules:

The equations (9) and (10) of the paper calculate the expected value and covariance of the distribution propagated through the dynamic model (2).

Solving:

A_k|k-1=E(A_{k-1|k-1}+Ts * u_{k-1}+Ts w_{k-1})

P_k|k-1 = Cov(A_{k-1|k-1}+Ts * u_{k-1}+Ts w_{k-1})

, where E(...) computes the expected value results in (9) and (10).

Dear Rashmiranjan Nayak

These links might be an asset, have a look:

1. https://www.sciencedirect.com/science/article/abs/pii/S0262885620302109?dgcid=rss_sd_all

2. https://arxiv.org/abs/2008.08944

3. https://dl.acm.org/doi/10.1145/3394171.3413973

Kind Regards

Hi Nick Brose

If you are interested in lysosomal distribution and localization, antibody staining (for example Lamp1is a good marker on lysosomal surface) is perfectly compatible with PFA fixation. You can fix your cells, perform an immunofluorescence for Lamp1 and store your preparation at 4ºC for as long as you want. As Tanoy Dutta was saying, cells are not alive so viability is not an issue. But in case you need to study lysosomal pH, you will need your cells alive indeed, so you won't have to fix them with PFA. There are available probes pH sensitive such as LysoSensor.

Hope this is useful!!

sorted by relevance | date

@tariq islam .Use dead reckoning.it is the process of calculating current position of some moving object by using a previously determined position, or fix, and then incorporating estimations of speed, heading direction, and course over elapsed time.

Can you use glycosylation to probe membrane protein topology? Fig. 1D in the following article discusses this.

Thank you so much.

Dear Dr. Yuqian Li

Perhaps by the time I am writing you this note you have seen this site. I

hope I have helped something

regards

Reinaldo

Dear Boxuan Simen Zhao

Thanks you for sharing the details.

You mentioned that the AAV particles have worked earlier - did it work for Neurexin for the same neurons that you are trying to target now? If yes, then I guess there might be some issue with the AAV production.

One common problem is the recombination events of the ITRs in the plasmid. This might compromise the expression of your target protein. I would highly recommend to perform a control digest for the AAV construct. You can find some other helpful tips for troubleshooting here - https://blog.addgene.org/tips-for-a-1st-time-aav-user-by-a-rookie-aav-user

Also, I came across this plasmid which supposed to have much better tropism for mouse brain - https://www.addgene.org/166921/ . Maybe this will be help to improve the results from your experiments.

Good luck !

Hey Shubham,

it depends on what questions you would like to answer with your experiments. I'm also not sure if i fully understand your question but maybe this helps:

1) -> it is always useful to read and know the important literature.

2) -> transcriptomic data and comparative genomic studies are useful to identify relevant genes for a certain issue. When it comes to transcriptomic data a useful approach is to expose your organism/cells to a certain stress to detect up-/downregulated genes compared to non exposed cells. Therefore it is important to design your own experiments to answer your individual questions.

Finding only homologous genes/proteins, you can use several bioinformatic databases (BLAST, UniProt...) in this case you should know your target genes.

4) -> characterizing already known genes of the same organism/cells is not useful. Why would you do that when it's already reported? But you can investigate homologous genes of another organism (not reported!) to check if comparable gene sets/proteins are involved in e.g. draught/starvation etc... Finding completely unknown genes and classify them to a cellular event is not that easy as you might think :)

good luck!

Check this paper:

Dear Layth As. Jasim,

I suggest you to see links and attached file on topic.

Best regards

I believe we used a VectorNav Vn-100 but mainly because we already had it available at our lab.

thanks

Hi Versha, this online software runs on different Algorithms, as different algorithms have different ways to predict and sort the data, sometimes the prediction changes.

CELLO uses a two-level Support Vector Machine system to assign localizations.

WoLF PSORT is an extension of the PSORT II program for protein subcellular location prediction.

Some like PSORT db - uses laboratory experimentation (ePSORTdb dataset) and computational predictions (cPSORTdb dataset) data

Hi Hyunbae Lee,

If you are going to place your GOI before the mGFP, the transit peptide from your GOI will direct the GFP to wherever it goes inside the cell.

In order to make the cloning easier, I would suggest you to use NcoI site to clone your GOI by using a method called ELIC that allows you to insert your fragment in sense cutting with only one enzyme. (Find attached the paper) In our lab we use DH5a strain and we also realized that homologous sequences around 15bp to the vector works perfectly.

Best

You may try GOSim or GOexpress package from conductor for annotation of your gene list.

Very interesting Mr. Mohamed-Mourad Lafifi. Thank you for sharing the files.

You may be able to download from ftp://ftp.biologie.ens.fr/pub/molbio/

When lysosomotropic agents are used such as astemizole or itraconazole, mTOR is detached from the lysosomal membrane and is inactivated. This is one of the mechanisms by which lysosomotropic agents have anti-cancer effects. Therefore, I presume that mTOR is functional only while attached to the lysosomal membrane.

Dear Sathishkumar Moorthy

There are few code available regarding this subject.

If you are interested in the learning aspect, this article is good:

Learning Vision-based Flight in Drone Swarms by Imitation

IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. ACCEPTED JULY, 2019

Another very good article - WITH CODE (at least should be released soon): a drone is following a target with an AprilTag - the target can be the considered as the leader.

Fast-Tracker: A Robust Aerial System for Tracking Agile Target in Cluttered Environments

https://arxiv.org/pdf/2011.03968v1.pdf

code:https://github.com/ZJU-FAST-Lab/Fast-tracker

Regarding source code, another place to look at is:

Multi-robot Systems (MRS) group at Czech Technical University in Prague

Please look at the video that may fit your need: https://www.youtube.com/watch?v=Ax3ONfo1hMA

Best

Based on my experiences on implementing NHC on real products, there are some challenges:

1 - Misalignment between vehicle body frame and IMU frame may be estimated, you can detect it when plotting the signals in the rear-wheel frame.

2 - If the vehicle turns drastically or jumps, NHC should be deactive.

3 - The measurement noise covariance matrix for NHC should be adaptively tuned.

You can have a look at Groves's book Principles of GNSS, Inertial, and Multisensor Integrated Navigation Systems.

Please the see the article given below, very useful for your

Image binarization focusing on objects

Regards

@Mohamed-Mourad Lafifi, thanks so much sir. I really appreciate it sir.

Hi,

there can be one more issue in this regard. The TAG protein will give signal when properly folded. Some times the TAGs added at the N-ter and some time C-ter of the protein of interest. See if some literature exists to show that the TAG will give the signal the way you have cloned. If not, try to change the orientation of the TAG

Unbound receptor should be cytosolic. The receptor binds to ligand that has been taken into the cytosol by the cell and translocates to the nucleas where it interacts with the mechanism that produces mRNA. A number of older papers have described this phenomenon. Just from memory I would lookup articles written by A. Poland and J. Knutson of the U.W. Madison (1980s or so). I think their classic review article was 1980 or so.

Thanks Abrar Ahmed and Mohammed Salih Mahdi ..... I checked the link, it is a great software. One more question I wanted to ask. Can it annotate Hand joints? All the tutorials and the videos that I found were concentrating on finding segmentation masks and bounding box for a particular object.

Hi Rajeev Vikram,

Stellaris has a software where you can design RNA FISH probes.

All you have to do is to make an account and paste the sequence of the lncRNA.

It will have the different parameters like masking level, probe length, etc. which you can control while designing your probes.

here is the link for:

hope it is helpful for you,

Good luck