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Why are red blood cells preferred to study the structure of plasma membrane?

Why are red blood cells preferred to study the structure of plasma membrane?


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If we wanted to study the structure of a plasma membrane, why are red blood cells a more attractive cell type to work with than other cell types such as liver cells or kidney cells?


Human RBCs are relatively simple in structure compared to the other cells in question; they contain no cell organelles (so far I knew; please correct me if there is any new theory) and therefore contain only one membrane.

RBCs do not have significant amount of extracellular matrix making them very easy to work only with the cell membrane. RBCs float in a fluidy medium (blood-plasma), so easy to collect, distribute in containers, keep into various solution. no need of maceration. Human RBCs are very uniform in shape and size, and do not divide. That makes it useful for various experiments like demonstrating plasmolysis and deplasmolysis as well as quantitative experiments on membrane biomolecules.

One classic example is Edwin Gorter and F. Grendel that first showed that cell membrane is bilayer; that is upon observation on RBC. They took known number of RBC, denatured the membrane, extracted the lipids and spread that as a lipid monolayer on water-air interface. They found that the monolayer is twice bigger than total surface area of taken RBC !*

Source:*The Cell/ Cooper/ 4th Edition/ASM-Press and Sinauer publication.


However this is not a reference-based answer, but these are the features more than enough to make me so lazy that I would choose only RBC and flyaway from any-other option.


The Red Blood Cell as a Gender-Associated Biomarker in Metabolic Syndrome: A Pilot Study

In the present pilot study (56 patients), some red blood cell parameters in samples from patients with metabolic syndrome and subclinical atherosclerosis, but without any sign of coronary artery disease, have been analyzed. The main goal of this work was to determine, in this preclinical state, new peripheral gender-associated bioindicators of possible diagnostic or prognostic value. In particular, three different “indicators” of red blood cell injury and aging have been evaluated: glycophorin A, CD47, and phosphatidylserine externalization. Interestingly, all these determinants appeared significantly modified and displayed gender differences. These findings could provide novel and useful hints in the research for gender-based real-time bioindicators in the progression of metabolic syndrome towards coronary artery disease. Further, more extensive studies are, however, necessary in order to validate these findings.

1. Introduction

Metabolic syndrome (MetS) is a cluster of risk factors for atherosclerosis, including insulin resistance, hypertension, glucose intolerance, hypertriglyceridemia, and low high-density lipoprotein-cholesterol (HDL-C) levels [1]. Affected patients have a significantly increased risk of developing atherosclerotic disease, diabetes, and cardiovascular disease (CVD). This is probably due to a blood hypercoagulability as well as to endothelial cell activation. It has been hypothesized that the hypercoagulability state could predispose patients to venous thromboembolism [2].

Several epidemiological studies, the Framingham, in particular [3], have investigated into the evolution of cardiovascular disease hypothesizing the presence of a gender difference in the pathogenetic and progression determinants detectable in men and women. For instance, women were found to outlive men and to experience fewer atherosclerotic cardiovascular events, with an incidence lagging behind that in men by 10 to 20 years [4]. This gap in incidence closes with advancing age, when CVD becomes the leading cause of death in women as well as in men [5, 6]. In consideration of the high incidence of morbidity and mortality, due to CVD, and of the paucity of well-established gender-associated markers, further studies focused at identifying novel bioindicators should be considered as mandatory.

On these bases, a pilot study has been conducted in a low number of patients with MetS of both sexes and subclinical atherosclerosis with the aim to identify innovative peripheral blood biomarker in this preclinical phase [7]. We focused our attention on the red blood cell (RBC) as a candidate possibly implicated in these pathologic conditions. RBCs are peculiar cells aimed at the delivery of oxygen and nitric oxide to the periphery and carbon dioxide to the lungs. In addition, they also exert, under physiological conditions, a scavenging activity towards reactive oxygen and nitrogen species often overproduced in morbidity states, for example, in inflamed tissues. Their deformability, essential for their circulation in small blood vessels, is an important prerequisite for such vascular “antioxidant” functions. Conversely, when the redox state of RBCs is altered, erythrocytes can turn out to be a source of reactive species, and, consequently, its typical structural and functional features are lost [8, 9]. Importantly, the oxidatively modified erythrocyte increases its aggregability and adhesiveness to the endothelium and to other blood cells, thus contributing to vascular damage. In addition, CVD risk factors, namely, insulin resistance, obesity, and hypertension, all share a common abnormal ion profile in RBCs. This might help to explain their frequent clinical coexistence. Specifically, it has been hypothesized that RBC intracellular pH levels are lower and inversely linked to both body mass index (BMI) and fasting insulin concentrations either in normotensive or hypertensive individuals. Moreover, ionic imbalance, for example, of intracellular potassium, magnesium, and calcium, can decrease intracellular pH levels also resulting in a reduced GSH/GSSG ratio [10]. In this work, three different putative “indicators” of RBC injury and aging have been evaluated: glycophorin A (GA), CD47, and phosphatidylserine (PS). The first is a glycoprotein that is widely expressed at the surface of RBC and is downregulated during senescence [11] the second, CD47, as for other cells, is an integrin-associated protein that acts as a “marker of self” [12] the third is a phospholipid localized to the inner leaflet of the plasma membrane, which is externalized to the outer leaflet during cell remodeling leading to cell death, for example, by eryptosis [13, 14]. Notably, it has been reported that phosphatidylserine-exposing RBCs may adhere to the vascular walls [14] and may interfere with microcirculation as it has been proposed to occur in the metabolic syndrome [9]. Importantly, GA loss, PS externalization (evaluated in terms of positivity to its ligand: annexin V), and reduced expression of CD47, respectively, have been reported as critical events responsible for the removal of RBCs at the end of their lifespan [15–17].

2. Patients and Methods

2.1. Study Population

The study population consisted of 56 ambulatory subjects with MetS (31 men and 25 women, aging 50–70 years) and 40 age-matched healthy donors (HDs) (22 men and 18 women). All patients and HDs were Caucasian. All study subjects underwent a complete cardiovascular evaluation which has included: history and physical examination, heart rate, blood pressure, fasting serum glucose fasting plasma lipids, Fibrinogen, CRP, comprehensive two-dimensional echocardiogram, carotid echo-color Doppler and exercise ECG testing. Healthy donors were identified on the basis of the absence of CVD risk factors and a completely normal CVD screening.

MetS was diagnosed according to the amended National Cholesterol Education Program’s Adult Treatment Panel III (ATP-III) Guidelines in individuals meeting three or more of the criteria reported elsewhere [1]. Healthy donors were identified on the basis of the absence of CVD risk factors and a completely normal CVD screening. We included in the study (i) patients with an increased (N1 mm) carotid intima-media thickness (IMT), but in the absence of known or suspected coronary artery disease (CAD), and (ii) only women in postmenopausal and without hormone replacement therapy. The patient characteristics have been reported in Table 1.

Patients with previous myocardial infarction, previous coronary artery by-pass graft, coronary angioplasty or positive exercise ECG testing, depression, inflammatory diseases, and ACEI treatment were excluded from the study. The nature and the purpose of the study were explained to all participants who gave their informed consent following the rules of good medical practice. This study was approved by the Institutional Review Board of “Sapienza” University of Rome (Italy). The investigation was conformed to the principles outlined in the Declaration of Helsinki.

2.2. Isolation of Erythrocytes

Human erythrocyte suspensions were prepared from fresh venous blood collected as previously reported [11].

2.3. Analysis of the Redox Balance in RBCs

For intracellular ROS production, RBCs (5 × 10 5 cells) were incubated in Hanks’ balanced salt solution (HBSS, pH 7.4) containing dihydrorhodamine 123 (DHR 123, Molecular Probes, USA). Intracellular content of reduced thiols was explored by using 5-chloromethylfluoresceindiacetate (CMFDA, Molecular Probes). Samples were then analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif, USA). The median values of fluorescence intensity histograms were used to provide semiquantitative evaluation of reduced thiols content and reactive oxygen species (ROS) production.

2.4. Evaluation of RBC Injury

Quantitative evaluation of RBCs with phosphatidylserine externalization [11] was performed by flow cytometry after double staining using FITC-conjugated annexin V and 0.05% Trypan blue for 10 min at room temperature and analyzed by fluorescence-activated cell sorting (FACS) in the FL3 channel to determine the percentage of dead cells.

2.5. Quantitative and Qualitative RBC Protein Analyses

For glycophorin A detection, RBCs were stained with anti-glycophorin A (Saint Louis, Mo, USA) monoclonal antibodies and subsequently incubated with anti-mouse IgG-fluorescein-linked whole antibodies (Sigma). For CD47, RBCs were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Sigma Chemical Co, Mo, USA), stained with monoclonal anti-CD47 (Santa Cruz Biotechnology, CA, USA), and subsequently incubated with antimouse IgG-fluorescein-linked whole antibody (Sigma). Finally, all the samples were analyzed with a FACScan flow cytometer or observed with a Nikon Microphot fluorescence microscope.

2.6. Morphometric Analyses

Whole blood from MetS patients and healthy donors was stripped on the slide, dried at room temperature, and observed by light or differential interference contrast (DIC) microscopy. Altered erythrocyte shape was evaluated by counting at least 500 cells (50 RBCs for each field at a magnification of 1500x) from MetS patients and healthy donors.

2.7. Statistical Analyses

Cytofluorimetric results were statistically analyzed by using the nonparametric Kolmogorov-Smirnov test using Cell Quest Software. A least 20,000 events were acquired. The median values of fluorescence intensity histograms were used to provide a semiquantitative analysis. Statistical analyses of collected data were performed by using Student’s t-test.

3. Results

3.1. Redox Balance

Considering that changes in the redox state can contribute to the loss of RBC structure and function [8], two important parameters have been analyzed. We measured the reactive oxygen species (ROS) production and the total thiol content (essentially referred to as reduced glutathione). However, no significant differences were detected in the ROS and total thiol production in RBCs from patients with MetS in comparison with that from healthy donors (Figures 1(a) and 1(b)). Furthermore, no gender differences were observed.


(a)
(b)
(a)
(b)

Structure of Plasma Membranes

The plasma membrane (also known as the cell membrane or cytoplasmic membrane) is a biological membrane that separates the interior of a cell from its outside environment.

The primary function of the plasma membrane is to protect the cell from its surroundings. Composed of a phospholipid bilayer with embedded proteins, the plasma membrane is selectively permeable to ions and organic molecules and regulates the movement of substances in and out of cells. Plasma membranes must be very flexible in order to allow certain cells, such as red blood cells and white blood cells, to change shape as they pass through narrow capillaries.

The plasma membrane also plays a role in anchoring the cytoskeleton to provide shape to the cell, and in attaching to the extracellular matrix and other cells to help group cells together to form tissues. The membrane also maintains the cell potential.

In short, if the cell is represented by a castle, the plasma membrane is the wall that provides structure for the buildings inside the wall, regulates which people leave and enter the castle, and conveys messages to and from neighboring castles. Just as a hole in the wall can be a disaster for the castle, a rupture in the plasma membrane causes the cell to lyse and die.

Figure (PageIndex<1>): The plasma membrane: The plasma membrane is composed of phospholipids and proteins that provide a barrier between the external environment and the cell, regulate the transportation of molecules across the membrane, and communicate with other cells via protein receptors.


Deformability

Deformability is the ability of a red blood cell to change shape when it squeezes through a small space, like a capillary. Capillaries can be as small as 3 micrometers (um) wide , while a healthy red blood cell is between 6 and 9 micrometers wide. Red blood cells must pass through openings narrower than they are, and that means red blood cells have to deform quite a bit to make it through these tiny passages!

Like red blood cell shape, deformability also depends, in part, on cell membrane composition. With the right fats, carbs, and proteins in the cell membrane, red blood cells should be able to deform to make their way even through the tiniest blood vessels. Without the right components in the cell membrane, however, the cell membrane can become stiff enough to slow blood flow, or even fragile enough to rupture under pressure.

Red blood cell shape and deformability may be able to help us understand issues with blood flow in ME / CFS, since both shape and deformability have an effect on blood flow. While there are older studies on blood flow and RBC deformability in ME / CFS, there are now new and more accurate technologies we can use to measure deformability and blood volume. Scientists at the Stanford Genome Technology Center and San Jose State are working together to develop a sensitive assay to measure RBC deformability by examining flow through artificial capillaries.

Using these new techniques, we can more closely examine blood flow to determine whether or not it is compromised in ME / CFS. Understanding why red blood cells in ME / CFS may have unusual shapes or compromised deformability may connect to the metabolic abnormalities we have seen in some studies . Understanding this aspect of ME / CFS pathology can help us learn more about the ME / CFS disease process as a whole, and lead to a diagnostic assay that clinicians can add to their diagnostic toolkit.

Other links you might be interested in:
History of ME / CFS
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Averting a second pandemic:

Open Medicine Foundation leads groundbreaking international study of

Long COVID’s conversion to ME/CFS

AGOURA HILLS, CALIF. — Open Medicine Foundation (OMF) is leading a large-scale international collaborative study investigating the potential conversion of Post-Acute Sequelae SARS-CoV-2 infection — more commonly known as Long COVID or Post-COVID Syndrome — to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a chronic, life-altering disease with no known cause, diagnostic test or FDA approved treatments available.

Up to 2.5 million people in the U.S. alone suffer from ME/CFS the COVID-19 pandemic could at least double that number. An estimated 35 percent of Americans who had COVID-19 have failed to fully recover several months after infection, prompting many to call it “a potential second pandemic.”

OMF recognized a familiar health crisis emerging, one with eerie similarities to ME/CFS. This crisis presented a unique opportunity to understand how a viral infection — in this case COVID-19 — may develop into ME/CFS in some patients. The goal is to find targeted treatments for ME/CFS patients and ultimately prevent its onset in people infected with SARS-CoV-2 or other infections.

The federal government is only now investing in Post-COVID research, with no focus on its connection to ME/CFS. OMF has already engaged researchers for the largest-scale study of its kind, solely supported by private donors who have contributed over one million dollars to date. When fully funded, the five million dollar, three-year study will be conducted across the globe at OMF funded Collaborative Research Centers, led by some of the world’s top researchers and ME/CFS experts.

In a significant percentage of patients, infections preceded their development of ME/CFS. For example, according to the CDC about one in ten infected with Epstein-Barr virus, Ross River virus, or Coxiella burnetti develop symptoms that meet the criteria for ME/CFS.

The ability to follow the development of ME/CFS from a known viral infection is unprecedented to date and crucial to researchers’ understanding of the disease. The focus of this study is to find the biological differences between persons returning to good health after COVID-19 and persons who remained ill more than six months after infection and developed ME/CFS. Understanding these alterations in key pathways can lead to groundbreaking discoveries including new biomarkers, drug targets, and prevention and treatment strategies.


Methods

The RBC membrane is the primary contributor for its mechanical nature since a RBC does not possess any internal structure. The lipid-bilayer contributes to its out-of-plane bending resistance and surface area incompressibility while the cytoskeletal spectrin network contributes to the in-plane shear deformation [53], and its cytoplasm contributes to the volume incompressibility of the RBC. The Helmholtz free energy of the RBC membrane is the collective contribution of out-of-plane membrane bending energy, in-plane shear energy and the energy penalty due to cell surface area and volume constraints relative to the specified reference membrane configuration. The equilibrium RBC shape is determined at the minimum free energy state of the RBC membrane.

Free energy of the CG-RBC membrane model

The developed CG-RBC membrane model employs particles to represent the actin junctional complexes of the RBC membrane and form a 2D triangulated surface of triangles. The adjacent particle-particle connections of the triangulated surface represent the cytoskeletal spectrin links. The in-plane shear energy of the RBC membrane is estimated based on the coarse-graining approach implemented by Fedesov et al. [50], and is composed of an attractive potential in the form of the WLC potential and a repulsive potential in the form of a power function. can be expressed as follow [50] (5) (6) (7) where, is the length of link, is the Boltzmann constant, is the absolute temperature, is the maximum link extension, is the persistence length, is the power function coefficient, and is an exponent such that is defined as . The experimentally estimated RBC membrane shear modulus lies between 4–12 μNm -1 [9, 50], and can be expressed as following for the CG-RBC membrane model [50]: (8) where, is the equilibrium length of spectrin link and defined as . The parameters and can be estimated for a given and using Eqs (5) and (8) at the equilibrium state of specified cytoskeletal reference state.

The out-of-plane bending energy of the RBC membrane is estimated based on a discrete approximation of the Helfrich energy model [54] for a zero spontaneous membrane curvature, such that: (9) is the membrane curvature at the triangle-pair that shares the link, and represents the surface area associated with the link. and can be given as: (10) (11) where is the angle between outward normal vectors to the triangles sharing link, and and are the planer area of and triangles respectively that share the link. The concave arrangement of a triangle-pair results in positive whereas the convex arrangement of a triangle-pair results in negative . An illustration of the triangle-pair made of and triangles that shares the link is provided in Fig 2.


Discussion

The purpose of this study was to determine the extent to which a variety of RBC-based fatty acid metrics were associated with incident T2DM over 11 years of follow-up. We originally examined five metrics, two of which, D5D and D6D, we found to be significantly related to risk for diabetes, the former inversely and the latter directly. The other three metrics (the omega-3 index, the PUFA factor, and LA) were not related in fully-adjusted models, with the exception that the omega-3 index was inversely associated with risk in women under 70 years of age. Further analysis of all individual RBC fatty acids revealed that palmitic and palmitoleic acids were directly related to risk for incident disease, the strongest of which was palmitic acid (see below).

Connections between fatty acids and diabetes have been explored in many settings with several different study designs (see review by Riserus et al. [30]). Since dietary intake surveys provide only a rough estimate of in vivo fatty acid status (stronger relations for some and weaker for other fatty acids [31]), the use of circulating fatty acid levels as biomarkers of status is preferred. [32] In the study of incident T2DM, biomarker-based studies have more commonly found significant relations with disease than diet survey-based approaches when compared in the same contexts. [6, 10–12, 17, 33]

Fourteen previous reports have been published on the relations between fatty acid biomarkers and incident T2DM (Table 1). Most took a discovery approach and examined a relatively full suite of fatty acids, whereas others use a hypothesis-based approach and focused on a few specific fatty acids. As is evident from the Table, the present study is the second largest to date (but far smaller than the EPIC-InterACT study [16]) and is the only one done nested in a randomized controlled trial for hormone therapy in postmenopausal women. The fatty acids or metrics most consistently reported to be adversely associated with incident diabetes across these studies are palmitoleic acid, palmitic acid, and the D6D ratio and to be favorably associated, LA and the D5D ratio. These, along with the omega-3 fatty acids, will be discussed below.

Desaturase ratios and related fatty acids (AA/DGLA and DGLA/LA)

Among the most consistent fatty acid metrics associated with risk for T2DM are the desaturase ratios. Of the six past studies that examined them, five found significant inverse relations with disease for the D5D ratio and/or direct relations for the D6D ratio [6, 7, 10, 12, 19] and the study that did not find these associations included only 30 events [14]. Consistent with these findings, Warensjo, et al. [34] found direct relations for D6D and inverse relations for D5D and the development of metabolic syndrome over 20 years. The extent to which these ratios actually reflect hepatic enzyme activities is unclear, nevertheless, as circulating fatty acid-based biomarkers, their relations with future T2DM appear to be robust. In intervention studies with fish oil, D6D was reduced and D5D was increased [35], but these ratios were unchanged by differences in total dietary fat [36]. The RBC D5D and D6D ratios are highly correlated (r = 0.68) which is not unexpected since DGLA is in the denominator in the former and the numerator of the latter. Indeed, DGLA alone [6, 7, 12] and LA alone [6, 7, 10, 12, 14, 37] were commonly associated with risk, and here we observed an association of DGLA with incident disease (p<0.01, although this did not meet the <0.002 criterion for multiple testing). AA alone was never found to be associated with risk for T2DM. What these findings imply is that factors that lower DGLA levels—either by enhancing conversion to AA or by slowing conversion from LA, may favorably influence metabolic pathways involved with the development of diabetes. Whether DGLA metabolites, such as the 1 series prostaglandins or other oxylipins [38], may have adverse effects (e.g., higher DGLA is associated with higher CRP and lower adiponectin levels [39]), or AA and/or LA metabolites favorable effects remains to be seen., Diets rich in LA (e.g., 14% en) lower levels of DGLA and AA in serum CE, whereas diets very low in LA (e.g., <2% en) increase levels of the longer-chain metabolites [40]. These fatty acid distributions may simply be epiphenomena caused by dysglycemic processes that incidentally up-regulate the FADS2 gene and at the same time down-regulate the FADS1 gene (which code for D6D and D5D, respectively). A recent exploration of the potential T2DM factors associated with the desaturase ratios in the EPIC study suggested that liver fat accumulation, but not high density lipoprotein cholesterol, adiponectin or C-reactive protein, may be mediating the relationship [41]. Regardless of the question of causality, the RBC D5D and D6D ratios have the potential to risk-stratify patients for T2DM.

Palmitic acid

RBC palmitic acid gave the strongest signal with incident T2DM of all the fatty acids examined here, confirming the findings of others [7, 10, 13, 16]. Although the third most prevalent fatty acid in the diet (about 20% of total), its levels in RBCs do not correlate well with intake [10, 12, 42] nor do they respond proportionally to changes in intake [42, 43].8 This is largely because palmitic acid is also an “endogenous” fatty acid synthesized de novo from the products of carbohydrate metabolism. Nevertheless, considerable evidence has accumulated that diets rich in saturated fatty acids (about 2/3 rds of which in the US come from palmitate) and carbohydrates and low in unsaturated fatty acids increase insulin resistance, perhaps via their up-regulation of lipogenic and suppression of fatty acid oxidative pathways (see review by Riserus [30]) which can lead to hepatic steatosis [44] and to metabolic syndrome [13]. Palmitate has also been shown to stimulate ceramide synthesis in skeletal muscle which increases tissue insulin resistance [45, 46].

Palmitoleic Acid

In contrast to trans palmitoleic (produced by ruminant bacteria and derived largely from dairy products [47]) which was inversely related to risk for incident T2DM in the Cardiovascular Health Study, [8] cis palmitoleic was directly associated with incident T2DM in our study, which derives mostly from hepatic and adipose synthesis. [48] It is increased with high-carbohydrate diets [36, 42] and a marker of de novo lipogenesis [49]. Palmitoleic was directly related to risk in 8 of the 9 previous studies in which it was examined. The present study confirms these findings. It had been hypothesized [9] that, based on considerations about the potential feedback inhibition of adipose-derived palmitoleic acid on hepatic lipogenesis, higher levels would be associated with improved glycemic status and reduced risk for T2DM, but as noted, this has not proven to be the case. Diets with a high ratio of polyunsaturated to saturated fatty acids (largely LA to palmitic) alters cell membrane biophysics [50], and improve binding of insulin to skeletal muscle nuclei and stimulate glucose transport [51].

Omega-3 fatty acids

As noted, overall the omega-3 index was not associated with incident T2DM, but in women under 70 (mean age of the cohort) it may be beneficial. Djousse et al. [11] and Virtanen et al. [15] are the only others to report an association between incident T2DM and long-chain omega-3 fatty acids (measured in plasma phospholipids or serum, respectively). The Cardiovascular Health Study cohort [11] was of similar age to ours and follow up of similar duration but contained 42% males how this may have affected these relations is not clear since sex was adjusted for in their model. EPA+DHA was not significantly associated with incident disease until low density lipoprotein cholesterol and plasma phospholipid LA levels were added to the model, and family history of T2DM and education were not included. We also confirmed an earlier observation [15] that docosapentaenoic acid (n-3), the metabolic intermediate between EPA and DHA, is beneficially associated with incident disease. Although this fatty acid may be storage form of EPA (by retroconversion), little is known about the physiological effects of this fatty acid, much less how it might be involved with diabetes 34. The inconsistency among biomarker-based studies regarding relations between omega-3 and T2DM is somewhat reflected in the heterogeneity seen among diet-based studies [52] where an increase of one serving of fish per week was associated with a 5% increased risk for T2DM in six US cohort studies, with no change in three, and with a 2% reduction in risk in five studies from Asia/Australia. Clearly, the relations between omega-3 fatty acid biomarkers and T2DM remains unclear.

Other fatty acids

No other fatty acids (besides palmitic and palmitoleic) were significantly associated with incident T2DM after adjusting for multiple testing. Two fatty acids with p-values <0.01 merit comment, however. DGLA (mentioned above) and myristic acid. The latter was directly associated with incident diabetes supporting the findings from a large European study [16]. Myristic is an endogenous fatty acid reflecting, along with palmitic and palmitoleic acids, de novo lipogenesis [53] with some arising from palmitate oxidation and/or laurate elongation [54]. In another study RBC myristic levels were marginally directly related to the risk of developing metabolic syndrome, they were not associated with incident T2DM [13].

Cross sectional relations of fatty acids with pre-diabetes markers

Differences in desaturase ratios in dysglycemic patients reported in cross sectional studies [39, 55–57, 40, 41] suggest that the disease process itself may be altering fatty acid metabolic pathways. Thus, a reverse causation situation could be occurring whereby patients with elevated D6D and/or depressed D5D ratios at baseline may already have “subclinical” T2DM. The loss of the significant relations between the desaturase ratios and incident T2DM when baseline hemoglobin A1c was included in the models in one study also supports this possibility [7]. In order to at least partially control for this, a sensitivity analysis was conducted in which we eliminated all incident cases of T2DM in the first two years after the RBC samples were collected. This did not alter the significant relations with these two ratios seen in the entire cohort suggesting that reverse causation was not at play here.

Certain limitations should be noted, such as unmeasured confounding. Also, the use of only a single measure of RBC FA content at baseline (instead of serial assessments over the 11 year period) reduced our ability to accurately describe the long-term exposure. Second, as noted above, owing to a misstep in sample processing, the RBC PUFAs (especially) were variably damaged and had to be reconstructed based on experimental degradation studies and multiple imputation techniques as described in Pottala et al. [14]. Third, as discussed above, there was likely some misclassification due to the use of self-report for diabetic status. All of these factors added variability to the assessment of both exposures and outcome, which may alter the observed associations in various directions. Finally, these results apply to elderly (mean age 70), postmenopausal women however, the robust findings associated with D5D and D6D ratios in racially and gender mixed populations suggests these results are generalizable to the general population (Table 1). The principal strengths of the study were its size, duration of follow-up, a well-characterized national cohort of women, an objective biomarker of fatty acid status, the evaluation of the full set of RBC fatty acids for relations with incident T2DM, and the inclusion of a sensitivity analysis to address potential reverse causation.

In conclusion, lower levels of RBC palmitic acid and the D6D ratio and higher levels of the D5D ratio were significantly and independently associated with incident T2DM over 11 years median follow-up in the WHIMS cohort. Whether there is a causal link between these fatty acid distributions and incident disease cannot be discerned from this study, but RBC fatty acid data may have value in stratifying patients for risk of T2DM.


The Diffusion of Water- Osmosis

Like carbon dioxide and oxygen, water is able to move across the cell membrane from areas of high concentration to low concentration. This movement is aided by the presence of small channels created by proteins, which are called aquaporins. When water diffuses across a membrane, it is referred to as osmosis 8 . Often, water will move across a membrane in order to balance the unequal concentrations of a solute, which is not able to move through the membrane itself. A solute is a material that is dissolved in a liquid. For example, in salt water, the solute is the salt.

Osmosis is most easily understood by imagining an experiment. Imagine a beaker that has been divided in half by a membrane that is permeable to water and impermeable to sugar, like most cell membranes in animals. Imagine that red sugar (for the purpose of this example, you will have to pretend that the sugar itself is actually red, so that as you add it to water, it turns light pink and the more you add the darker red the water gets) has been added to side A and very little sugar has been added to side B. Nature wants things at equilibrium, and there is not a state of equilibrium between the two sides of the beaker. At this point, side A should be dark red and side B would be a very light shade of pink. The simplest solution to the non-equilibrium problem would be for the sugar to move across the membrane until half of the sugar molecules are on side B and half are on side A. However, the membrane separating the two sides does not allow sugar to pass. An alternate solution to the non-equilibrium problem is possible. Water molecules can move from one side to the other to even out the concentration of sugar and water on both sides. If one side of the beaker (side A) is red and the other side (side B) is light pink, as it has very little sugar, then equilibrium could be achieved when both sides A and B are an equal shade of pink. This can happen when water passes through the membrane from side B into side A until the solute concentration of both sides was the same. (Notice that this would, of course, decrease the volume of side B and increase the volume of side A.) Once again, this does not mean that the water would simply stop moving once both sides turned pink. It means that water would be moving at an equal rate between sides A and B. (If this unit were designed for a high school class or even a high level middle school, it would be appropriate to discuss concentration and osmotic pressure in detail, but for the unit being proposed, the descriptors "higher concentration" and "lower concentration" will suffice. )

Osmosis&mdashand the need to regulate solute concentrations in organisms, or maintain homeostasis&mdashcan be demonstrated by placing cells in solutions of varying solute concentrations. Cells already have a certain level of dissolved solutes within them. If a cell is surrounded by a solution with the same level of solute concentration as the cell itself, there will be no net movement of water into or out of the cell. This solution would be referred to as an isotonic solution, meaning the concentration of solutes inside the cell and outside of the cell were the same 9 .

If the same cell were surrounded by a solution that had more dissolved solutes than the cell itself, then water would leave the cell in order to reach equilibrium. Think back to the hypothetical red sugar example above. The end goal is pink, and water will flow from where the color is a darker red to where it is lighter or where there is no color at all. The darker red would be outside of the cell and the lighter pink would be inside of the cell. This would cause the volume of the cell to decrease as water left the cell. In this case, the solution surrounding the cell would be described as a hypertonic solution.

If the same cell were instead placed in distilled water or water with very few solute molecules, then water would enter the cell in an effort to reach equilibrium. This would cause the cell to expand in volume. If the concentration difference was large, so that a large volume of water had to move into the cell to equalize concentrations, this process could actually result in the cell bursting. In this case, the external solution would be described as a hypotonic solution. Because most cells have a relatively large number of solutes in their cytoplasm, maintaining an environment that is isotonic to the cell is imperative. For the paramecium, a single-celled protist usually found in freshwater, there is a constant struggle to remove water that flows into the cell in a futile effort to reach equilibrium. In order to prevent the paramecium from rupturing, the paramecium has a contractile vacuole which continually pumps the water out of organism 1 0 .


Lab 1 Osmosis

Cells have kinetic energy. This causes the molecules of the cell to move around and bump into each other. Diffusion is one result of this molecular movement. Diffusion is the random movement of molecules from an area of higher concentration to areas of lower concentration. Osmosis is a special kind of diffusion where water moves through a selectively permeable membrane (a membrane that only allows certain molecules to diffuse though). Diffusion or osmosis occurs until dynamic equilibrium has been reached. This is the point where the concentrations in both areas are equal and no net movement will occur from one area to another.
If two solutions have the same solute concentration, the solutions are said to be isotonic. If the solutions differ in concentration, the area with the higher solute concentration is hypertonic and the area with the lower solute concentration is hypotonic. Since a hypotonic solution contains a higher level of solute, it has a high solute potential and low water potential. This is because water potential and solute potential are inversely proportional. A hypotonic solution would have a high water potential and a low solute potential. An isotonic solution would have equal solute and water potentials. Water potential (y) is composed of two main things, a physical pressure component, pressure potential (yp), and the effects of solutes, solute potential (ys). A formula to show this relationship is y = yp + ys. Water will always move from areas of high water potential to areas of low water potential.
The force of water in a cell against its plasma membrane causes the cell to have turgor pressure, which helps maintain the shape of the cell. When water moves out of a cell, the cell will loose turgor pressure along with water potential. Turgor pressure of a plant cell is usually attained while in a hypotonic solution. The loss of water and turgor pressure while a cell is in a hypertonic solution is called plasmolysis.
Hypothesis:
During these experiments, it will be proven that diffusion and osmosis occur between solutions of different concentrations until dynamic equilibrium is reached, affecting the cell by causing plasmolysis or increased turgor pressure during the process.
Materials:
Lab 1A – To begin Lab 1A, first collect the desired equipment. The materials needed are dialysis tubing, Iodine Potassium Iodide (IKI) solution, 15% glucose/ 1% starch solution, glucose Testape or Lugol’s solution, distilled water, and a 250-mL beaker.
Lab 1B – For Lab 1B you will need to collect six presoaked dialysis tubing strips, distilled water 0.2M, 0.4M, 0.6M, 0.8M, and a 1.0M sucrose solution six 250-mL beakers or cups, and a scale.
Lab 1C – Lab 1C these items are needed: a potato, knife, potato core borers, six different solutions, and a scale.
Lab 1D – During Lab 1D, only paper, pencil, and a calculator will be needed to make the calculations.
Lab 1E – n Lab 1E these items are needed: a microscope slide, cover slip, onion cells, light microscope, and a 15% NaCl solution.
Procedures:
Lab 1A – After gathering the materials, pour glucose/starch solution into dialysis tubing and close the bag. Test the solution for presence of glucose. Test the beaker of distilled water and IKI for presence of glucose. Put the dialysis bag into the beaker and let stand for 30 minutes. When time is up test both the bag and the beaker for presence of glucose. Record all data in table.
Lab 1B – Obtain the six strips of dialysis tubing and fill each with a solution of a different molarity. Mass each bag. Put each bag into a beaker of distilled water and let stand for half and hour. After 30 minutes is up, remove each bag and determine its mass. Record all data in its appropriate table.
Lab 1C – sing the potato core borer, obtain 24 cylindrical slices of potato, four for each cup. Determine the mass of the four cylinders. Immerse four cylinders into each of the six beakers or cups. Let stand overnight. After time is up, remove the cores from the sucrose solutions and mass them. Record all data in its appropriate table.
Lab 1D – Using the paper, pencil, and calculator collected, determine solute potentials of the solutions and answer the questions asked to better understand this particular part of the lab.
Lab 1E – Using the materials gathers, prepare a wet mount slide of the epidermis of an onion. Draw what you see of the onion cell under the microscope. Add several drops of the NaCl solution to the slide. Now draw the appearance of the cell.
Data:
Lab 1A – Table 1.1

Contents Initial Color Final Color Initial Presence of Glucose Final Presence of Glucose
Bag 15% Glucose/ 1% Starch Solution clear Dark blue + +
Beaker H2O+IKI Orange to brown Orange to brown _ +

Lab 1A Questions
1) Glucose is leaving the bag and Iodine-Potassium-Iodide is entering the bag. The change in color of the contents of the bag and the presence of glucose in the bag prove this.
2) In the results, the IKI moved from the beaker to the bag, this caused the change in the color of the bag. The IKI moved into the bag to make the concentrations outside the bag equal to inside the bag. The glucose solution moved out of the bag making glucose present in the beaker. The glucose moved to make the solute concentration inside and out of the bag equal.
3) If the initial and final percent concentration of glucose and IKI for in the bag and the beaker were given, they would show the differences and prove the movement of these substances to reach dynamic equilibrium.
4) Based on my observations, the smallest substance was the IKI molecule, then the glucose molecules, water molecules, membrane pore, and then the starch molecules being the largest.
5) If the experiment started with glucose and IKI inside the bag and starch in the beaker, the glucose and IKI would move out of the bag to make the concentrations equal, but the starch could not move into the bag because its molecules are too big to pass through the semipermeable membrane.

Lab 1B Table 1.2 Dialysis Bag Results


We would like to thank Marie Olsinova, Daniela Glatzova, Tony Magee, Martin Hof, Lukasz Cwiklik, Piotr Jurkiewicz, and Tomas Chum for critical discussions which led to writing of this article.

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Keywords: plasma membrane, membrane organization models, nanodomains, heterogenous distribution, membrane physical properties

Citation: Bernardino de la Serna J, Schütz GJ, Eggeling C and Cebecauer M (2016) There Is No Simple Model of the Plasma Membrane Organization. Front. Cell Dev. Biol. 4:106. doi: 10.3389/fcell.2016.00106

Received: 13 June 2016 Accepted: 14 September 2016
Published: 29 September 2016.

Manuel Jose Prieto, Instituto Superior Tຜnico, Portugal

Dylan Myers Owen, University of New South Wales, Australia
Richard M. Epand, McMaster University, Canada

Copyright © 2016 Bernardino de la Serna, Schütz, Eggeling and Cebecauer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


RESULTS

Characterization of a Duffy-specific antibody for the identification of cell types that express gp-Fy.The murine monoclonal antibody, anti-Fy6,26,27 yielded inadequate labeling for immunoelectron microscopic studies (not shown). Of the several rabbit polyclonal antibodies that we developed, antibody 6615 was preferred because it yielded the proper resolution for ultrastructural localization of the Duffy antigen (see below). Although the other rabbit polyclonal antibodies were robust in immunoblots, they yielded a very weak signal in fixed tissues, possibly due to the hindrance of the antigenic sites (not shown).

Antibody 6615 was generated with performic acid and CNBr-treated gp-Fy and did not react with deglycosylated gp-Fy (Fig 1). It appears that the performic acid treatment, which is the most effective procedure to disassemble the polymeric form of gp-Fy21 and CNBr cleavage, had converted the carbohydrate component of gp-Fy into a potent immunogen. The lack of immunoreactivity with erythrocyte ghosts of Duffy-negative individuals and deglycosylated gp-Fy, proved the immunological and chemical specificities of antibody 6615, respectively (Figs 1 and 2).


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