Science topic

Cholesterol - Science topic

The principal sterol of all higher animals, distributed in body tissues, especially the brain and spinal cord, and in animal fats and oils.
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Since it is not possible to utilize radioactive anymore, I would like to know if there are other validated ways to perform cholesterol absorption assay in vivo in mice.
Thank you.
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you can use biomarker techniques which is the same as scintigraphy and see the cholesterol pathway
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standard diet for normal groups, and high-fat diet for the rest groups. From the references I read, the normal cholesterol for rats was around 50-100 mg/dL, but the normal rats without any high-fat diet given have cholesterol level above 100 mg/dL. Does anyone have reference about the cholesterol and hypercholesterol value level for rats?
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normal blood cholesterol level is 10-54 mg/dl (Smith and Mangkoewidjojo 1998). The paper is https://media.neliti.com/media/publications/220603-blood-cholesterol-levels-of-hypercholest.pdf
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I am studying the interaction of a peptide with components of a membrane. One of the components, for example, cholesterol, is present throughout the membrane. What I want to do is to calculate the distance between the center of a particular amino acid like Phe (at 4th position) and center of the Chol molecule that is present closest to the amino acid.
I used gmx make_ndx to isolate indices of the aa residue and all the chol molecules, however, the indices of the cholesterol molecules are all clustered together without any separation in the text file, hence when I use gmx distance with the select flag, I get the center of mass of the whole system of cholesterol molecules and not the COM of each molecule. This is the command line I used: (I renamed the groups)
gmx distance -f md_trial2.xtc -s md_trial2.gro -n 4PHE_With_Headgroups.ndx -oav avgdist_4PHE.xvg -select 'com of group "4PHE" plus com of group "Chol"'
This works but of course gives me the wrong result.
One option that I have is to manually create index groups for EACH chol molecule and then calculate distances for each of them using a similar command, but there are 96 cholesterol molecules so to do it manually every time would be a daunting task, hence it would be of a huge help to me if you could help streamline this process.
Also, I mentioned Chol as a representative example; I am mainly focusing on headgroups of gangliosides in the membrane.
Thanking you for your help.
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All cholesterol molecules are considered, that is the strength of the RDF method it allows you to compared the positional relationship between a single molecule and a group molecules. This is especially useful for bilayers where the components can laterally diffuse around, so the peptide might not interact with the same molecules throughout the entire simulation.
You cannot identify the individual molecules in the selection group as the RDF is based on the overall density and not the distinct molecules.
So if you need to know which specific cholesterol molecule is the closest because you need to select it for another analysis you cannot use this method, but if you are looking to see if there is some interaction between the residue and cholesterol that causes them to prefer to be at a specific distance from each other this method takes into account all the cholesterol molecules over the full simulation period.
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Just curious if measuring glycemic and cholesterol parameters in mice after fasting them for 6 hours gives more accurate read -outs than fed state.
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Fasting gives more accurate results for both glycemic and cholesterol examination.
But if you want to check glucose tolerance then perform fasting test first then go for random after glucose intake
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I am trying to adapt the protocol found below, but they use THP1 cells, I am using murine BMDM. I am wondering if this is possible? I cannot find purified mouse HDL or apolipoprotein anywhere and we would like to avoid purifying it ourselves if possible.
Cholesterol Efflux protocol I am trying to adapt:
To assess NBD-cholesterol efflux, macrophages were incubated in phenol red-free RPMI 1640 medium containing 5 µmol/l NBD-cholesterol for 4 h at 37°C. Following incubation, the cells were washed with PBS three times and were then incubated with HDL or apoA-1, as lipid acceptors. To determine the correlation, various concentrations of HDL (5, 20, 50 and 100 µg/ml) and apoA-1 (10, 20, 50 and 100 µg/ml) were used for the [3H]-cholesterol and NBD-cholesterol efflux assays. HDL concentrations ranged between 5 and 100 µg/ml, and apoA-1 ranged between 10 and 100 µg/ml. Subsequently, the cells were harvested after 4 h, and the medium and cell lysate were collected for the detection of FI. Triton X-100 (0.1%) was used to lyse the cells in a 12-well plate, and the cells lysate was homogenized by pipetting up and down several times. A total volume of 600 µl was then aliquoted into three wells of a 96-well plate for the measurement of FI. The percentage of NBD-cholesterol efflux was calculated by dividing the FI in the medium by the sum of the FI in the medium and cell lysate. All data were from three independent experiments, each performed in triplicate.
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Ikemefuna Ikesee limited in what way?
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my project work based on cholesterol reducing property of lactic acid bacteria. I have cholesterol powder but it is soluble in organic solvent. but organic solvent will affect my bacterial growth. please suggest any method to dissolve cholesterol.
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Hello Sayli,
as a lipophilic molecule, pure cholesterol is more or less insoluble in water. You are not the first researcher facing this problem. For some potentially useful hints, you might want to have a look at the answers given to the following closely related questions which have been asked earlier on RG:
Can anyone suggest how to make cholesterol to soluble in water?
(16 answers)
and
Can anyone suggest any cholesterol dissolving solvents?
(23 answers)
As you will see, some answers are helpful and some are not. In any case it is often a good idea to use RG directly as a valuable source of information.
Good luck with your work!
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I´m currently working on testing the effects of certain food extracts and their effect on the in vitro micellar solubility of cholesterol. I've been trying to emulsify the cholesterol with taurocholic acid but the cholesterol doesn't even interact with it. I'm using this reactive (https://www.sigmaaldrich.com/MX/es/product/sigma/c8667). I'm thinking about changing to water-soluble cholesterol, but I want to hear your experiences working in this assay or with cholesterol in general.
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Hi Zabdiel,
I'm also currently doing the similar work as yours. Have you tried sonicating the solution? I sonicated the solution and it was quite homogeneous.
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Hi there,
Are you aware of any good European lab focused on lipidomics of metabolites of Cholesterol synthesis pathway?
Thanks a lot in advance!
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For what it is worth, no guidelines will ever get predictions right until they use lipid ratios.
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Health impacts of saturated fat seems to be contentious. Reports showing negative effects are numerous but inferences of some reports showing positive effects cannot be neglected at all. Based upon latest findings and summing up them with past research conclusions, what can be the best suggestion for common people today ?
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The following RG link is also very good:
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I am planning to make cholesterol assimilation test for my pure lactic acid bacteria cultures. However, i have a water-insoluble cholesterol. So, how can i dissolve this in mrs agar and also not give to any harmness to incubate my cultures
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Reply to Mohamedelfatieh Ismael : Is it reasonable to do so? After all, ethanol may affects the bioactivity of bacteria Mohamed Elfatieh Ismael
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Long term side effects of anti cholesterol drugs?
While we can control the cholesterol by some nutrients!
Please share your own idea, not copy and paste the media
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According to the patient's condition with cholesterol, if it is chronic, there are no side effects.. because leaving the treatment will increase the level of cholesterol in the blood and have more side effects.. Best regards
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I am planning to make cholesterol assimilation properties of my pure cultures. However, I have a water-insoluble cholesterol. So, what is the best medium to dissolve my cholesterol. It is important because, I am desired to use this cholesterol to add MRS broth medium. So, for example, if I dissolved my cholesterol in ethanol, it may have negative effect on microbial growing. So, I need a medium for my cholesterol that have no harm to my microorganisms also.
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Hi Caglar, you can find detailed information about the assimilation of cholesterol in broth here in this paper.
H. Aloǧlu and Z. Öner, “Assimilation of cholesterol in broth, cream, and butter by probiotic bacteria,” European Journal of Lipid Science and Technology, vol. 108, no. 9, pp. 709–713, 2006
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Whether the pH of lysis solutions (while RNA extraction) need to be modified as per the cholesterol content in the cell's plasma membrane?
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Saurabh Tiwari Always welcome
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Thanks to anyone how would like to spare me attention first. I am working on treating primary mouse hepatocyte with drugs may cause dyslipidemia, hoping to find any positive results on their influences. I have a master question as, what kind of treating medium should I choose? Currently I am applying William E with CM 4000 supplement for both maintaining and treating, while I feel like that too much supplements may have unexpected influence on the outcomes. However, I also wonder that applying poor medium like William E only would strongly damage primary hepatocyte functioning, which may also cause unwished results. May anyone with experiences help me with this?
Besides, I also need a positive control to validate both the SREBP pathway upregulation and cholesterol increasing. Statins can upregulated the SREBPs and Hmgcr by blocking their activity, causing cholesterol lowering, which is not ideal for me. May anyone also have ideas about this can help?
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interested
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I am treating cells with water-soluble cholesterol. (https://www.sigmaaldrich.com/SG/en/product/sigma/c4951) Most papers recommend 1-10mM. Upon treatment of cells in medium, even within 10 minutes, I can see crystallization of the cholesterol.
How should I treat my cells with the cholesterol?
Thank you.
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Dear Alice Chan this seems to be a common problem when working with so-called "water-soluble cholesterol". Unfortunately I'm not a specialist enough to give you a qualified answer. However, as a chemist I might add that "water-soluble cholesterol" is in reality the inclusion complex cholesterol-methyl-β-cyclodextrin. As such it is not just a mixture of the two components, but the cholesterol is encapsulated in the cavity of the cyclodextrin. I'm pretty sure that the formation of this inclusion complex is reversible depending on factors like solvents and temperature. Thus I assume that it is advisable to always use freshly prepared solutions.
It might also be worth having a look at the answers given to the following closely related RG questions:
How to store water-soluble cholesterol?
(3 answers)
and
How to complex cholesterol in B-cyclodextrin for better solubility and stability?
(9 answers)
Good luck with your work and best wishes!
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I am facing problem in cholesterol assimilation protocol for human nutrition project
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As increase in level of cholesterol in the body is one of the major factor responsible for heart attack, because deposition of which block the arteries and cause the obstruction in the blood flow. As cinnamon powder has several medicinal values but is any scientific report showing the reduction of cholesterol in the body ?
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Have a look at this useful RG link.
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I'm using methyl-β-cyclodextrin (MBCD) to deplete cholesterol from mammalian cells. I'm following the Mahammad and Parmyrd protocol, in which cells are treated with different MBCD concentrations, ranging from 0.5 mM to 2.5 mM to evaluate the level of cholesterol depletion. However, in the protocol description, they use 20 million cells in one ml, and add one ml of the desired concentration (0.5 mM to 2.5 mM of MBCD). Although the protocol advises not to deplete cholesterol in adherent culture, sometimes is needed depending on the experiment, as I have seen many authors do, but, the reported concentration in those papers is usually 5mM in adherent cultures as if they put a 5mM MBCD solution in medium in the plate or well regardless the cell number. Should I normalize to the cell number in my well or plate? or just pour a 5mM solution on the plate without considering the number of cells?
Thanks in advance
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Dear Carolina, thank you for posting this interesting technical question on RG. Unfortunately I cannot provide you with a qualified expert answers because we are inorganic chemists. However, I can suggest to you the following relevant research article which might help you in your analysis:
Cholesterol Depletion Using Methyl-β-cyclodextrin
This paper is freely available as public full text on RG. It has a detailed experimental section. Also please have a look at the following potentially useful article:
Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies
This paper has not yet been posted as public full text on RG, but both authors have RG profiles. Thus you can easily request the full text directly from one of the authors via RG.
Good luck with your research and best wishes, Frank Edelmann
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Dyslipidaemia is one of the well established modifiable cardiovascular disease (CVD) risk factors. Elevated levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and to a lesser extent triglycerides are associated with an increased risk of atherosclerotic CVD and its clinical end points, Coronary Heart Disease, Cerebrovascular Disease and Peripheral Arterial Disease. In contrary, Elevated levels of high-density lipoprotein cholesterol (HDL-C) are inversely correlated with CVD risk. This is attributed to the anti-atherogenic role of HDL by removing cholesterol from the tissues to the liver "Reverse cholesterol transport" for further metabolism and excretion via bile. What are the possible mechanisms to augment the anti-atherogenic effect of HDL?
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Interested
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Dyslipidemia like elevated serum cholesterol and reduced high density lipoprotein is associated with erectile dysfunction.. Is this dyslipidemia the cause or the effect of erectile dysfunction?
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Please also see the RG link below.
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I have Martini coarse-grained model of protein in complex with cholesterol. I have followed the backmapping tutorial (http://md.chem.rug.nl/index.php/tutorials-general-introduction-gmx5/others-gmx5#Backward). Now, I am trying to backmap my system (protein-cholesterol), it does not work. It works for proteins only.
I am using the charm36 forcefield, and the initram-v5 script for backmapping.
For cholesterol it says:
Checking dependencies:
backward.py ... Missing dependency: backward.py
Thanks in advance
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You have to make backward.py executable with the command "chmod +x <fileName>".
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One of my relative admitted in hospital. he had angioplasty. Interventional Cardiologist recommended him to take medicine Ramipril BP (Controls Blood pressure) and Rosuvastatin (Controls cholesterol) for his entire life. My question is If a cardiac patient control the daily diet (lowers intake of salt and avoid cholesterol containing food) is it still necessary to use Ramipril BP and Rosuvastatin? Thanks in advance
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Yes, he needs to continue those drugs in addition to diet and life sthle medication. Drugs, lifestyle modifications and risk factors control all together may give ardsult equivalent to bypas surgery or sngioasty in patients with stable coronary hesrt disease.
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Angioplasty is common problem today. If it is possible to deliver efficient drugs to the exact target, it may be a new era in cardiovascular field of treatment...
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The recommended dietary allowance (RDA) for B3 is only 18 mg a day, far less than the amount needed to improve cholesterol levels so effective targeted drug delivery may be helpful.
If anyone already suffering from artery blockage then there should be some way other than surgery and ballooning.
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We read that people who breathe a lot of dust or asbestos can suffer from fibrosis in their lungs. I have seen that some people also suffer COPD without that and wondered if it has ever been related to high cholesterol? ie. Can fibrosis of the lungs be created from multiple tiny pulmonary embolisms over time when someone has high cholesterol?
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I want better repeatability of my protocol and this is hindered by the fact I have to weight each phospholipids everytime i make a new batch which leads to slight changes and errors. So I would prefer to take out those lipids out of a solution instead. I will use the lipids dissolved in ethanol anyway so it would be really convenient.
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These should be stable if stored in a freezer (-20oC) and flush the air out using N2 to reduce any oxidation if you have unsaturated fatty acids as components of your lipids. I sometimes use antioxidants (BHT) also if these do not interfer with your assays. Good luck
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Can anyone help me with measuring cholesterol and triglyceride levels in zebrafish embryos?
Is there any specific end point measuring kit available?
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Fathima,
do you have access to a department for clinical chemistry? They could run your samples on their machines.
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Experts have also found that restricting food intake during the day can help prevent health problems such as high cholesterol, heart disease and obesity, as well as improve mental health and well-being. Apr 20, 2020 (SOURCE: AL JAZEERA).
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Dear Fatema Miah,
Assalamalaicum.
Ramadan fasting has got benefical effect on mood, memory and attitude.
And as you can remember 'Autophagy and fasting' it has got to do lot with mental function including Alzehmer.
But patients with major pschychiatric illness may need to be prohibited from fasting, same rule as it is applicable to other physical ailments.
There is recent study showing, during Ramadan there is less no of admission observed in psychiatric wards.
Much Dua for you and your sons.
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I just came across premade NGM (Nematode Growth Medium). It is a powder mix but has Cholesterol to be dissolved with water, then autoclave. My lab always uses the original recipe. We always add Cholesterol after autoclave.
Is Cholesterol autoclavable?
The powder formula I saw is:
Agar 17.5 g/l Sodium Chloride 3.0 g/l Peptone 2.5 g/l Cholesterol 0.005 g/l
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Wow. Interest question
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hi,everyone:
I try to analysis cholesterol concentration in serum and liver recently , I see protocol and it says that the serum lipid extract sovlent do not need to add triton, but liver do.
I am curiously about why there are different?
Also, l analysis triglycerides concentration in serum and liver, both do not need to add triton.
l can’t understand why triglycerides do not need to add triton?
sorry for my poor grammar, i hope you guy s can understand what i mean 😢
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Dear Ting,
For plasma TG and Chol , you dont need triton x-100
however for liver lipid extraction , you need a so called Bligh&Dyer extraction.
After the extraction TG and Chol are in a chloroform phase , the tritonx100 2% is needed to resolve the lipid in a water phase .
Lipid in water phase are used in the comercial kits
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This PUFA stock solution will be use to treat cells.
Is it the same preparation than for cholesterol (50 mg Cholesterol + 5 ml Ethanol 99.5%)?
Thanks in advance for your help.
Tony
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Do you want to add them as non-esterified fatty acids (NEFA) ? You can then try turning them into "soaps" by adding just enough potassium hydroxide (one potassium atom per fatty acid molecule). Then mix with bovine serum albumin (final concentration 0,2 to 0,5%; it's a lot) to keep them solubilized and to protect the cells from the detergent effect of fatty acids.
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Dear all,
can somebody help me concerning the preparation of a cholesterol solution for cell culture ? Do I have to prepare a stock solution in ethanol and then dilute it in culture medium at a ratio of 1:1000 ? Any other ideas or possibilities ?
Thanks in advance. Tony
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Thank you all for your answers !
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We are planning to add cholesterol to cells in order to raise the intracellular cholesterol concentration and confirm the protein expression of SREBP2. I didn't know if I should dissolve cholesterol in a water-soluble solution, so I asked a question. I would appreciate it if you could teach me how to dissolve cholesterol.
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Hello. I have a dataset containing three groups: low cholesterol, medium cholesterol and high cholesterol. Each group has about twenty participants. For each participant I have their mean nocturnal blood gluocse level for 4-6 different days. I would ideally like to compare mean nocturnal blood glucose levels between the three different cholesterol groups. I'm aware that unless I take a mean nocturnal blood glucose across the 4-6 days for each participant and then compare these values then using a one way ANOVA would violate the assumption of independant samples, but doing this would lose a lot of useful data. Is there a way that I can include all of the participant's individuals nights in a comparison between the cholesterol groups? Many thanks
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you could also do a 2-way RM ANOVA, to take the different sample collection times into account. If you do not have repeated measure for each participant, a mixed model would be best.
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So i have this intriguing idea why dont we make hdl and inject to the patients not in a regular basis, but it will be just as dialysis for cholesterol. Of course it will be taking iv, but actually taking iv saline is like taking water in egypt so why dont we make it more benificial and add hdl to it
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As I understand your question, you wish to suggest using HDL for patients with dyslipidemia, with the ultimate aim to reduce atherosclerosis and prevent the related cardiovascular disease (CVD) risk.
HDL's ability to reduce atherosclerotic plaque depends largely upon its capacity to remove cholesterol from the plaques in addition to several other properties that you have listed above.
The manufactured (recombinant) HDL has shown promise in experimental conditions and animal studies. However, its efficacy in humans is yet to be tested. Even other HDL-elevating therapies have not shown much effect due to the HDL remodeling that occurs in dyslipidemic individuals.
However, it appears that recombinant HDL has the potential to become an important arm of future combination therapies aimed at the reduction of atherosclerotic plaque and CVD risk.
References
1. Ibanez B, Giannarelli C, Cimmino G, et al. Recombinant HDL(Milano) exerts greater anti-inflammatory and plaque stabilizing properties than HDL (wild-type). Atherosclerosis. 2012;220:72-7.
2. Ben-Aicha S, Casaní L, Muñoz-García N, et al. HDL (High-Density Lipoprotein) Remodeling and Magnetic Resonance Imaging-Assessed Atherosclerotic Plaque Burden: Study in a Preclinical Experimental Model. Arterioscler Thromb Vasc Biol. 2020;40:2481-2493.
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I am trying to use water-soluble cholesterol (Sigma C4951) in cell culture. Does anyone know whether I can store this item after dissolving in water, or does it need to be made fresh every time? The product sheet only says that the powder needs to be stored at -20.
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Hello Yi Jing
I suggest you prepare fresh solution each time. However, if you have experiments queued up one after another you can store the extra solution at 2°C to 8°C for a maximum of one week. But try using the stored solution at the earliest.
Thank you.
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I did a liposomal preparation of DPPC:CHOL 1:1 with flow ratio 2:1 (PBS: Ethanol) based on literature of review using microfluidics nano-assembler bench top. I meaured the size using Malvern zetasizer and it was 100nm ( which was expected) however I got very high zeta potential for triplicates (-7330, -7840, -8040mv) any ideas why this happened. size is good but zeta is extremely high.
I repeated the experiment, changed the concentration, checked for contamination. filteration ...etc though it's the same.
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We know the reason for the very high values - it's nothing to do with the sample. It's good old fashioned operator error. Unfortunately, even after three requests from people willing to help, the original questioner has not posted an example .dts file so that we can resolve the problem.
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hi
I am trying to simulate electroporation for a bilayer containing 10% cholesterol on GROMACS. I have read lots of articles but the only parameter that they have talked about was the electric field magnitude. So I am not sure about the other parameters like angular frequency, time at of the peak in the field strength and width of the pulse.
Is there any references that could help me with that?
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thanks Sourav Biswas for your answer, but my main problem is electric field properties for electroporation phenomena in a bilayer.
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l've got rat serum samples with different background (from pink to yellow) and l'm trying to test total cholesterol, HDL and triglyceride in mice serum. l'm worried that the background noise would still exist even l diluted samples. Are there any alternatives to solve this problem? Can l spin serum again (10min, 4c, 1500rcf) before testing? Glad to hear your suggestions!
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There's two things I can think of, but it depends on your assay whether they're relevant.
If your colorimetric assay consists of two (or more) reagents, and the color is `developed` by the last reagent, you could measure 600nm OD immediately prior to adding the last reagent. This may be relevant if the triglyceride kit measures TGs indirectly via an assessment of glycerol. The TG kit which I used previously, and which is clinically used, performs a 'glycerol blanking' step, to be able to distinguish endogenous glycerol from the TG glycerol. In this blanking step, you are essentially also subtracting the background.
If that is not the case, and you have sufficient amount of plasma, you could prepare a buffer similar in composition to the coloring reagent, and measure the 600nm OD of the diluted plasma as is.
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In a physiology textbook treatment of reproduction, they describe the Corpus Luteum as yellow as a result of the color of cholesterol. Yet, pure cholesterol is white, and has no structures that would lend themselves to color in the visible spectrum. Also, cholesterol deposits in the body, not to mention egg yolks, are clearly yellow. So what is the basis of the yellow color?
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Thanks so much for that. I now do recall reading about the carotenoids in egg yolks, and it is interesting that the same is true of the corpus luteum. That is the common yellow color.
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hi.
I have done electrostatic potential analysis on a symmetric bilayer contains 10% Cholesterol but I've got this figure and I thought there is a problem with that as the potential is not symmetric and it is not equal in the both leaflets.
I will be thankful if you could help me solve this problem.
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Wojciech Kopec
thank you so much.
I just did not know about "-correct" option.
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When walnuts is good for reducing LDL and total cholestrol levels in blood,I think we can boil folium layer in kernel of Walnuts in water and use it as a usefull Liquid.
thanks.
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All the nuts, including Walnut are rich in polyunsaturated fatty acids, and reduce total cholesterol and LDL cholesterol.
Although leaves (folium) of walnut tree have been shown to have salutary effect on lipids, no study as such has used the folium of the kernal of the walnut
Have a look at this article in the link:
The Comparative Effects of Aqueous Extract of Walnut ...
3.
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Sulfa drug increase the the cholesterol and low density lipoprotein.. What is the mechanism of action of this drug which increase these types of lipid...
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Effect of Sulfa Drugs on Lipid Profile
1. Sulphonylurea group of antidiabetics increase Triglycerides compared with Insulin in diabetic patients.
2. Thiazide group of diuretics, mainly used in patients with hypertension, increase the TG, LDL-C, and VLDL-C in a dose dependent manner, although Indapamide is devoid of these dyslipidemic effects of other thiazides. (2-5)
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VLDLr play important role in cholesterol uptake, metabolism of lipoproteins and neuronal migration
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One does not need to know TG levels to predict the population at risk of atherothrombotic disease (ATD). TG are not much present--if at all--in ATD plaque. Once one accounts for HDL-c levels, TG wash out as an ATD risk factor. For the prediction of the population at risk of ATD, see my publication in EC Cardiology a few months ago.
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can anybody help me with In-vivo screening strategy for microbes having Bsh activity? I want to explore them for cholesterol lowering activity?
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I need fundus images of an eye with cholesterol related abnormalities..
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Hi.
I'm new to GROMACS and I want to simulate a DMPC bilayer contains 8 Cholesterol molecules. But I have no idea about how to add Cholesterol molecules to my simulation box.
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Hi,
Thanks
Wojciech Kopec
I will.
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I have determined part of (A) of a two part question where (A): the amount of cholesterol in your sample by reference to the standard curve, which is given to us. I am now unable to determine part (B) of said question which states I should:
Convert that (A) into the total cholesterol content of your 1g sample. CAREFUL you will need to take into account the fraction of the whole sample that you have loaded onto the HPLC.
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Hi, Lisa,
I think the problem is solved this way:
From the peak area curve, also called calibration curve, you can determine the concentration of your analyte cholesterol. I think you have already done this. So you have the value mg/kg or any other concentration. But let us stay with mg/kg.
From this concentration you can then simply determine the amount of substance in 1g or ml
Example: Concetration 10 mg/l = 10mg / 1000ml = 10/1000 mg /ml= 0.010 mg/l
At least I understand the question to mean that you should specify the absolute amount of cholesterol in your sample
I hope this has helped you.
With kind regards
Joachim
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Hi Dear friend,
Along as I am working on an animal model study as C57lb/6 mice to determining the cholesterol concentration in blood serum and in the brain
But now I am planning to extract the brain and the purpose wants to measure the cholesterol concentration. and if anybody know the procedure please recommend me or give me some idea about it?
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Hi everyone,
I am struggling with visualisation of possible deposition of cholesterol crystals in tissues of gastrointestinal tract.
I am wondering what method tu use, as there is a staining with filipin but from what I undestood - this will stain ALL OF FREE cholesterol (so also this deposited in cells), and I am not sure if that will make crystals vivible.
Second option - is using simply polarized light microscope, and here I would like to ask if anyone has experience or protocol he could share?
best!
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I think you may be right with polarization, cross Polaris might help with visualisation. Do you already have access to a polar scope?
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Welcom,
I need to check the amount of glucose and cholesterol in zebrafish blood. I have standard kit's from Sigma. My question is, schould I dilut the blood before or not. There is nothing in protocol abouth how to prepare zebrafish blood.
Kate
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This protocol may also help you.
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Hi!
I am searching for a method of visualisation/staining protocol for cholesterol crystals (not a free cholesterol, just crystalized) in cells as well as tissues?
anyone has any experience??
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  • V. S. Borovik et al., Visualization during Crystallization in Minkowski Spacetime V.S. Borovik, V.V. Borovik and D.A. Skorobogatchenko Solid State Phenomena in the year 2017 Vol.269. PP7-14. DOI: 10.4028/www.scientific.net/SSP.269.7(Scopus)
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Three week ago, bmj rapid response team publish announcement (Link: https://www.bmj.com/content/368/bmj.m1182/rr-10) This rapid response "as you see" tell us statin like cholesterol lowering drugs may be worsen covid 19 infection. In this research area's (cytokine level, infection rate and statin molecular pathways or etc..) literature very poor. So in this situation born few questions:
1. We use statins reduce cytokine levels. Most of covid 19 case had a severe cytokine storm. So, cut off patient's statins make higher rate of covid 19 infection mortality?
2. A lot of research show high risk for infection people who have a low cholesterol level.
What are your thoughts? Anyone have a molecular and clinical link for statin and covid19?
Thanks.
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The following are the reasons for why the High Fat diet does not increases the cholesterol-
  • Marked reduction in cholesterol absorption - the mechanism for this effect is not known
  • Greatly increased synthesis of bile acids - the patient synthesized roughly twice the mass of bile acids as control subjects
  • Reduced endogenous cholestrol synthesis (Kern F: Normal plasma cholesterol in an 88-year-old who eats 25 eggs a day. New Eng J Med 324:986, 1991.).
An increase in the amount of cholesterol ingested each day increases the plasma concentration slightly. However, when cholesterol is ingested, the rising concentration of cholesterol inhibits the most essential enzyme for endogenous synthesis of cholesterol, thus providing an intrinsic feedback control system to prevent an excessive increase. As a result the plasma conc. usually not altered more than ±15 percent by altering the amount of cholesterol in the diet, although the response of individuals differs markedly. A high saturated fat diet increases blood cholesterol conc. 15 to 25 %. Ingestion of diet containing highly unsaturated fatty acids usually depresses the blood cholesterol conc. a slight to moderate amount (Guyton’s physiology).
The study conducted by R B Nair et al, revealed that even after 3 hour of consumption of oil/ghee, lipid profile was not increased, instead there was a decrease in lipid levels after 6 days of Snehana therapy.
Vasant et.al, (Jamnagar, 2006) in their study on standardization of Snehana observed that in group A, triglyceride & VLDL levels were increased significantly after Virecana may be due to Avara Śuddhi. In group B, triglyceride & VLDL levels were increased significantly after Snehana but after Virecana, both have came to normal level, due to the proper Śodhana. So from these evidences, it can be concluded that, after proper Snehana, one has to do proper Śodhana, otherwise lipid levels may be raised and in case of known subjects of obesity & hyperlipidemia, one should have to perform Rukshaṇa Karma before Snehana to avoid the increase of lipids.
Even though the above data shows the safety of use of Snehana therapy, one has to do multi-centered trials on large number of patients with scientific research methodology. Then only myth about Snehana can be cleared from the modern fat conscious world.
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Does higher proportion of cholesterol (3%) in high fat diet is affects the feeding behavior of Sprague dawley Rats? Thanks in advance ?
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Thank you Ishraq Jasim
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we did plasma measurement of cho and tri. in our mouse samples, now we would like to measure direct level in the fresh tissues, does anyone know where we could do this around montreal area, or any kits that can be used for this!
thanks
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Hi Dear friend,
Along as I am working on an animal model study as C57lb/6J mice to determining the cholesterol concentration in blood serum and in the liver.
But now I am planning to extract the liver and the purpose wants to measure the cholesterol concentration. and if anybody know the procedure please recommend me or give me some idea about it.
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I am performing a screen to evaluate the role of lipoproteins/cholesterol on gene expression, in which using lipid reduced/free FBS is essential. I have previously used Hyclone Lipid Reduced FBS SH30855.02 but unfortunately Thermo Fisher has discontinued this item. Any suggestions on substitutes from a reliable source? Thank you!
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Hi there,
maybe it is too late , but our company , Purmabiologics makes the highest quality of Lipid depleted FBS
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After going through the literature only virgin olive oil looks like help to increase HDL. However, olive contained high amount of omega-9 than omega-3 which may induce inflammation. Despite much hype, there is no good literature to suggest that coconut oil is good for increasing HDL. It increases cholesterol level significantly due to high saturated fatty acids. Flaxseed oil too fail to increase HDL.
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Please have a look at the following RG link.
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I am working on Mice and want to make hypercholesterolemia. But after giving cholesterol powder the level of cholesterol has decreased from previous level. Could anyone please suggest me how will I make hypercholesterolemia in Mice rather giving cholesterol powder?
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De novo synthesis of cholesterol enhanced by insulin therefore if you give only cholesterol it is regulated, to increase lipogenesis give high doses of both cholesterol and glucose for long time.
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How do I select only the phosphate group from my phospholipids and oxygen from cholesterol and then club all the phosphates and oxygens into one group in an index file? Kindly help.
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Try this:
  • Open the gro/pdb file to identify the names of the atoms you are interested in. For example, let's say these are O7, P8, O9, O10, and O11 (the PO4 group from an all-atom phospholipid)
  • grep -Ew "O7|P8|O9|O10|O11" file.gro | awk '{print $3}' > index.ndx
  • (if it's a PDB, it will be '{print $2}')
  • Open index.ndx and add a group name such as [ phosphate_atoms ]
The basic idea here is to use grep to grab the atoms you are interested in, and then use awk to get the column of interest. Using this way, you should be able to get the phosphate + cholesterol oxygen at the same time.
There are multiple ways to do this, this is just one such way.
Hope that helps,
John
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I need to calculate the concentration of Total cholesterol in rat tissue samples. can anyone provide me a colorimetric assay without using any kits
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Adam B Shapiro W.J. Colonna Thanks a lot for your inputs. Really appreciate it
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I want to know about the working principle behind lipid profile test.
How it works.(in detail)
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Thanks everyone.
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Hi everyone
i plan to measure the cholesterol concentration in the culture medium to see how much cholesterol is taken up by the cell. But the medium has Phenol Red and it may affects the result? i wonder how to solve that, maybe i can set a blanket as the base? but i don't know how to calculate. the analysis machine i use is Qubit® 2.0 Fluorometer.
Thanks.
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Is there a relationship between the serum cholesterol level in poultry and the proportion of cholesterol in poultry meat?
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let me be more accurate,
Is increased cholesterol in the blood of birds must be followed by increased cholesterol in bird meat?
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Hi All,
I am novice in free energy calculation with metadynamics. I tried to calculate the free energy (using the attached plumed.dat file) of a biolocal multicomplex system containing lipids, cholesterol, and protein in the form of monolayers separated by a layer of water. The whole system is placed in a priodic box and a NP is placed above the monolayer. The NP freely moves towards the monolayer during the simulation with plumed. Keeping the similarities with the following paper
I wanna calculate the perpendicular distance between the NP and the monolayer surface. Each monolayer contains
NP 1
Protein_A 4
DPPC 244
POPG 104
CHOL 32
What will be the approximate density contour, width of Gausian, height of the Gausian?
I have fine-tuned the plumed input parameters (please see plumed.dat) e.g., the Gausian width, height, and Baisfactor. After 10 ns run with the NP freely interact with the monolayer lipids-proteins, the simulation suddenly stopped with an error (please see Error.txt). I am simulating the system using Martini force field.
Please suggest me about the things that I need in these situation.
Thanks in advace.
Regards
Sheikh Imamul
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The parameters for metadynamics simulations require some tests. You should run several simulations with different parameters and check whether the outcome is dependent on the parameters. For instance, use smaller width of Gaussian potentials, try lower frequency of putting gaussian, and employ smaller biasfactor.
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I would like to stain slides prepared from biopsies of GI tract for cholesterol (and cholesterol crystals), would you recommend any filipin staining protocol ?
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Hi Michal,
There is a filipin (compound similar to digitoxin) method for free cholesterol.
Fixation and sections: cryostat sections, post-fixed; frozen sections of fixed tissue.
Stock solution: 2.5 mg filipin in 1 ml dimethylformamide.
Staining solution: 0.2 ml stock solution in 10 ml PBS.
Method: wash sections in PBS.
Stain for 30 min in filipin solution.
Wash in PBS (x2).
Mount in PBS or glycerine jelly.
Examine by fluorescence microscopy (excitation BG12 with 512 nam barrier filter).
Results: Free cholesterol shows strong silvery fluorescence.
original method by Kruth & Vaughan 1980 - quoted in Bancroft & Stephens histology manual.
Good luck with your project.
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I am looking for a fluorescent probe or stain that would only bind surface cholesterol and that it is suitable for sections of fixed tissue.
I think filipin can be used cells, but I am not sure if it works with tissue.
A protocol would be appreciated.
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I've got an intractable issue with loss (apparently) of sterol acetates (cholesterol acetate, stigmasterol acetate, etc) when trying to quantify them via GC-FID. I routinely get 60-70% of what I should be observing. I can observe this both when acetylating standard sterols or when measuring a directly made-up cholesterol acetate standard. My measurement of my standards isn't a problem, as I see the same 30% deficit (for cholesterol) when comparing an acetylated sterol standard with a silylated one (i.e. the TMS derivative gives me what I think I should see, the acetate derivative doesn't). This isn't happening to n-alkyl alcohols, and it's not dependent on retention time (later eluting, non-sterols like archaeol acetate show up just fine), suggesting it's not an injection discrimination problem.
I see a baseline drop after my sterol acetates peak, which does not occur with underivatized sterols or TMS-ethers, or any other class of OH-acetate derivative. The shape of the baseline around the peak would seem to indicate decomposition of the compound on the GC column. However, the problem persists when a brand new column is used, or when a different phase is used (we have used both VF-17 and DB-5 columns, mid-polarity and non-polar, respectively), and there's no obvious reason why these compounds, which are generally quite stable, would be decomposing mid-flight. We've tried reducing the max oven temperature, changing flow rates, varying inlet temperatures, two different GCs, etc. Still keep getting ~70% response. We've tried this with both a PTV inlet and a SSL inlet. We're injecting in toluene. I added a retention gap to reduce interaction with the phase while the compound was 'waiting' to volatilize in the temp program, but this didn't change anything.
Any thoughts? Has anyone ever run into something like this? Or know why an unsaturated sterol ester would decompose on a GC column? I assumed there could be reactions with silanol groups, but the consistency of that that 70% regardless of column age or run parameters is extremely vexing.
I know there are corrections that can be made for the differential FID response of different types of molecule (in this case, a sterol acetate vs my n-alkane response factor standard) for the MOST precise quantification, and I've used simple mass balance corrections or carbon number corrections when comparing acetate and TMS derivatives, but I've never had a problem comparing alkanes and esters to a 1st order approximation before, certainly not to the tune of a 30% difference in response. Thoughts?
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John Canham Actually... I guess the Sylon is only going to help with the tubing, as everything else is metal. Still, couldn't hurt I suppose.
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Dear all,
I am new to GROMACS and I am trying to understand how to properly build and run a simulation of a lipid bilayer. In particular I am trying to build a lipid bilayer made of three kind of lipids: a free fatty acid (lignoceric), a cholesterol and a ceramide (cer2).
I downloaded the structure files and the force field from ATb (GROMOS 54 a7). Then, following the workflow of Justin Lemkul's second tutorial, I built my lipid bilayer system and run the first equilibrium stages. The problem is the following: once I start my unconstrained NPT production, ceramides and fatty acids stay in their position while many cholesterol molecules leave the lipid bilayer and move "inside" the bilayer, so in between the two leaflets. I know that it is possible that some cholesterol molecules can do this thing, but in my system this happens to too many of them (~30/40%). Honestly it seems really unreasonable and that's why I am here: did any of you have the same problem? Is it related to some kind of bad initial structure?
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Thank you for the answers. I used CHARM-GUI and the simulations are stable with a reasonable behavior of all lipid molecules. The only thing is that density profiles are quite different, so probably I'll just ask to the authors their topology/mdp/itp/etc files!
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I am looking for a simple and low-cost method of monitoring the cholesterol content in the cell membranes. My initial idea was to stain them with cholera toxin (anti-GM1 ganglioside) to get information about lipid rafts, so indirectly about the cholesterol content.
Do you have any better ideas?
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You have different options. The presence of cholesterol in the membrane gives rigidity, while the outflow of cholesterol causes membrane fluidity. Thus, the measurement of membrane fluidity can be an indirect measure of cholesterol levels in your cells. Another option is the use of fillipin. I leave you some links that I hope will serve you.
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Both PCSK9 inhibitors ans Statins are very powerful LDL-C lowering drugs. To start with, the JUPITER trial reported a 25% increase in NOD with rosuvastatin 20 mg, over a median follow-up of 1.9 years, compared to placebo and since then, several meta-analyses have confirmed a smaller but significant increase with various statins . Several mechanisms have been postulated underlying the derangement in glucose metabolism by statins. There is some evidence for the detrimental effects of statins on both insulin sensitivity and β cell secretion including the effects of increased influx of cholesterol due to inhibition of HMG-CoA-mediated intracellular cholesterol synthesis, inhibition of CoQ 10 and β cell apoptosis. Also one hypothesis states that lower levels of LDL-C can interfere with optimal β cell function. PCSK9 inhibitors are more powerful LDL-C lowering agents, then why only statins have been implicated in causing NOD and most of the studies done with PCSK9 inhibitors show no association. Is it for some commercial gain to make PCSK9 inhibitors, more popular LDL-C lowering agents?
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As you have rightly pointed the possible commercial explanation as the statin have now became generic and there is a need for one patented drug for business. This could be one of the factor. The same story of pioglitazine causing urinary bladder cancer created fear in prescribing physician and to change over to gliptins even though gliptins side effects or adverse events were more.
In order to prove it is better than available statins they willfully avoided few inclusion criteria in selected studies.
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I have seen several cases of low growth on children (girls) with no hormonal problem, but high blood cholesterol. Thinking about the similarity of the chemical structure between cholesterol and vitamin D, could there be some kind of causality?
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High LDL content - It is alarming for metabolism of Children as well as adult .
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Lately I find many cases of low growth in girls (3-4 years old) who also have high blood cholesterol. Could there be a relationship? I can't find studies that associate these two factors and the similarity of the chemical structure with vitamin D is what comes to mind.
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I'm using 1X 3b-Hydroxy-5-cholestene 5-Cholesten-3b-ol from Himedia for the culture of Helicobacter pylori ATCC49503 strain in Brucella broth, but unable to find any growth even after eight days. People have earlier successfully used 1X cholesterol from Gibco for such type of culture. Both products are cell culture grade. I'm wondering are they two different types of cholesterol and do bacteria prefer a specific form of cholesterol for their growth.
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H. pylori. is a fastidious organism capable of growing only under a narrow set of conditions. It must be cultured on nutrient-rich agars with whole blood from any one of a variety of large mammals such as horse or sheep. That asked and presented research input is to find out the oleaginous capacity of H. pylori. So subculture colonies are enough to carry out this research and must have followed safety label 2 during isolation and condign isolates. Now research initials in described form refers to determine oil capacity range in H. pylori ether it is cholesterol or not. First reported data should be positive when the oleaginous stained result comes out as it oleaginous, than next step is to be followed for determining nature of oil that either it contains saturated or unsaturated fatty acid. Alignment of results are to be followed and screened to find out correlation of this oleaginous activity on related with producing LDL or HDL. After extraction of oil and determination of nature, the methodology will be very easier to screen out for type of cholesterol by biochemical (Both organic and inorganic) test. Best of luck brother, this is my suggestions that i learned. Thank you.
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A few changes in one's diet can reduce cholesterol and improve heart health.
Kindly Share: Tips to reduce cholesterol, research findings, or anything related to the topic!!
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Dear Haghi A.K. - awesome point! Thank you for your reply!!!
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One of the reviewers of our manuscript said the following about the effects of cholesterol efflux on plasma cholesterol levels, but no references were provided:
There are several previous papers analyzing the effect of peripheral cholesterol efflux on plasma cholesterol levels and concluding that since the RCT pathway is very dynamic cholesterol removed via HDL particles is rapidly esterified via LCAT function and further taken up by the liver and this would not affect plasma cholesterol levels. Only in those cases where for instance liver SRBI receptor or LDLr/LRP-1 receptors are dysfunctional there might be slight effect via RCT on plasma total cholesterol.
If anyone has any further information or reference about this, I would highly appreciate it!
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Do not know if these papers are of any use to you:
Clinical efficacy and safety of achieving very low LDL-cholesterol concentrations with the PCSK9 inhibitor evolocumab: a prespecified secondary analysis of the FOURIER trial. Giugliano RP et al. Lancet. (2017)
Pleiotropic Effects of Statins on the Cardiovascular System. Oesterle A et al. Circ Res. (2017)
Overexpression and deletion of phospholipid transfer protein reduce HDL mass and cholesterol efflux capacity but not macrophage reverse cholesterol transport. Kuwano T et al. J Lipid Res. (2017)
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Does anyone know if the lipid levels in mice are comparable to humans ? What is the best method to measure LDL/HDL choleterol and TG in mice ? In advance, thank you for your help.
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why glibenclamide is associated with increases in triglycerides of streptozotocin diabetes-induced rats (male SD rats)?
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We are measuring total cholesterol levels secreted into the media by cultured astrocytes using the Abcam cholesterol assay kit (ab65390).
Because the number of cells differ between wells, and the hemocytometer is not very accurate, we are trying to normalize the total cholesterol values.
It was suggest that measuring total protein from the cells themselves, and normalizing: total cholesterol/total protein would be a possible idea.
The premise is that the more protein you have, the more cells there are.
Does this make sense?
Are there other suggestions?
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Yes, what you suggest is feasible. An alternate approach would be to stain your cells (left in the wells after harvesting the sups) with a nuclear stain like Hoechst 33342 and then read the fluorescence using a plate reader. The signal would be proportional to the number of cells you have in the well. If you rinse the cells with PBS before adding the staining solution, you may get rid of any dead/floating cells that may confound your results. The technique should take less time (<30 min) and effort. You can then normalize total cholesterol/Hoechst signal.