Question of the Day: Liquid Mechanics Involving Protein Powder

WHY DOESN'T THIS EVER MIX WELL IN WATER?!?!

Headache01 asks "Why doesn't powder mixed into drinks (like whey protein powders or cocoa) dissolve very well? It clumps into balls that are wet on the outside but remain dry on the inside. How can I make my whey mix better?"

Well, given that proteins, and especially mixture of proteins, are amphiphilic (meaning the molecule has a part with an affinity for water, and another for fat/oils), they tend to orient themselves into microscopic structures known as micelles. Micelles allow the molecules to isolate their lipophilic ends away from the water and their hydrophilic end towards water, ultimately forming a spherical, tubular, or sheetlike structure.

While micelles are a microscopic phenomena, a similar thing is what causes the clumps in your protein drink; along the protein powder-water interface there will be two-layer sheet type structures where the hydrophilic parts of the molecules are facing the water and the lipophilic parts stay away from it.

Wonder why you should avoid warm water when mixing powders? The heat causes the proteins to lose their secondary structure and become entangled with one another, making it difficult to break up the clump since the clump's outer surface has essentially polymerized. Thus, using cold water keeps the proteins tightly coiled and less likely to get entangled with each other.

I almost forgot! The solution is to wet each of the solid particles individually first before dispersing them (e.g. mix in a small amount of water to form a paste). This will ensure that they disperse well.

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Flow Cytometry In A Nutshell

FACS was used in the trial treatments of leukemia last Saturday's post.

Fluorescence-activated cell sorting analysis, or FACS, uses a flow cytometer to separate individual cells in a heterogenous suspension based on epitope type. Fluorochrome-labeled antibodies are added to the cell sample. The antibodies bind to specific epitopes on or within the cell. The fluidics system delivers a stream of cells or particles one at a time through an interrogation point, where it passes through a laser. A cell traveling through the laser beam scatters the beam’s light forwards and sideways as a function of its size and granularity, respectively. When the laser beam strikes cells labeled with fluorescent-labeled antibodies, the fluorescent dye becomes excited and fluoresces at a unique wavelength. The intensity of the scattered and fluorescent light is collected and filtered by the optics system, recorded by the detector which translates the light into a quantifiable electrical impulse that can be represented graphically as a dot plot or a histogram.

We can use different fluorochromes at the same time, as long as their emission peaks are far enough apart for us to easily distinguish. A peripheral computer can instantaneously analyze the forward and side scatter light and fluorescence to identify the characteristics of individual cells and separate them into different subpopulations by charging each droplet with either a negative or positive charge, depending on the intensity and wavelength of fluorescence, as they leave the stream. The droplet is deflected either to the right or left by charged electrodes into one of three sample tubes. Intensity and wavelength of fluorescence and be plotted in a two dimensional box plot, where subpopulations in the sample can be distinguished by looking at two parameters. 

2D results with two different parameters makes visualizing cell populations so easy!

A histogram measuring the frequency of celled labeled with antibody A is plotted on the y-axis of the two-parameter box plot, and another histogram measuring the frequency of cells labeled with antibody B is plotted on the x-axis. The box plot in this example shows two subpopulations, distinguished by the intensity of fluorescence f protein expression.

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New Cure for Leukemia?

Leukemia is the cancer of white blood cells.

The Penn scientists targeted chroniclymphocytic leukemia (CLL) by hacking a harmless version of the HIV virus to hack T cells in order to kill cancer cells. In previous studies, the cancer-killing cells died out quickly after infusion, but in this study, the genetically engineered cells multiplied a thousand-fold and were sustained for over 4 months. 

Let's go over the study first.  Three patients with chemotherapy resistant tumors had their blood drawn, separated, modified, and cultured. These patients underwent lymphodepleting chemotherapy, and their blood was reinjected. Endpoint assays were conducted a month after reinjection.

They did the same thing in mice, and the cells of interest were sustained for over six months, although I'm not sure whether the same monthly cycle was repeated. It doesn't say. But the cells of interest reached levels of up to 95% of white blood cells, up from 2.3-4.46% (figure 2). After an initial decay with first-order kinetics, the CART19 cell numbers stabilized between three to six months after reinjection. The fact that the cell levels were sustained after four months is at least some evidence the body can remanufacture the CART19 cells on their own.

What is most remarkable, however, is that the cells of interest seem to be able to remanufacture themselves within the body. In the third patient, flow cytometry showed that there were CAR19-expressing T cells with an absence of B cells 169 days after infusion. This is remarkable, since, "previous studies have not demonstrated robust expansion, prolonged persistence, or functional expression of CARs on T cells after infusion."

Figure 2 from the study showing levels of CART18 cells after infusion. Click to enlarge.

"There were no significant toxicities observed during the 4 days after the infusion in any patient other than transient febrile reactions. However, all patients subsequently developed significant clinical and laboratory toxicities between days 7 and 21 after the first infusion...With the exception of B cell aplasia, these toxicities were short-term and reversible. Of the three patients treated to date, there are two complete responses and one partial response lasting greater than 8 months after CART19 infusion according to standard criteria." The only side effect these three patients suffered was fever. One was hospitalized for a week, and another went into remission for 10 months.

In fact, "one of the preclinical rationales for developing CAR+ T cells with 4-1BB signaling domains was a projected reduced propensity to trigger IL-2 and tumor necrosis factor–α (TNF-α) secretion compared to CAR+ T cells with CD28 signaling domains (7); indeed, elevated amounts of soluble IL-2 and TNF-α were not detected in the serum of the patients." The cells infused into the patients were designed specifically to avoid a cytokine storm and to circumvent the donor's immune system.

"In our preclinical studies, we found that large tumors could be ablated and that the infusion of 2.2 × 107 CAR T cells could eradicate tumors composed of 1 × 10^9 cells, for an in vivo effector-to-target (E/T) ratio of 1:42 in humanized mice (8), although these calculations did not take into account the expansion of T cells after injection." In mice studies, billion cell tumors were ablated.

The three human patients had trillion cell tumors weighing around 1 kg before the infusion of CART19 cells. They all showed great progress, with the third patient surpassing others by 40:1. "Using the estimate of initial total tumor burden (1.3 × 1012 CLL cells) and the observation that no CLL cells were detectable after treatment, we achieved a marked 1:93,000 E/T ratio. By similar calculations, an effective E/T ratio in vivo of 1:2200 and 1:1000 was calculated for UPN 01 and 02 (table S6). Therefore, a contribution of serial killing by CART19 cells combined with in vivo CART19 expansion of >1000-fold likely contributed to the powerful antileukemic effects mediated by CART19 cells."

The trials for the three patients were financed by Alliance for Cancer Gene Therapy.

Source:  Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia

(the study) and MSNBC.

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No Science Sunday: GMSoccerPicks

Best Soccer News and Write Ups. Ever.

GMSoccerPicks is a must-read blog for you soccer fans out there. He's got some real insight as an avid soccer fan, and his commentaries should not be missed. The blog is updated daily, so if you're into soccer, bookmark it!


You can also join the hundreds of people following @gmsoccerpicks on Twitter and get soccer news as it happens, or like GMSoccerPicks on Facebook to get soccer write-ups in your newsfeed.

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Enumerating Bacteria In Lab

Serial dilutions allow us to do viable cell counts or total cell counts.

Serial dilution and plating can determine the amount of viable cells in a culture. Serial dilutions allow a discrete number of colonies of bacteria to grow, whereas concentrated cultures may contain billions of bacteria per milliliter. In serial dilutions, smaller dilutions are repeated in succession, and the dilutions can be multiplied to obtain the total dilution. Thus, serial dilutions are more practical than doing the total dilution in a single time. For example, if I have a 100 ml bacterial culture, I can add 1 ml of it to 99 ml of water, add 1 ml of the first dilution to 99 ml of water, and then add 1 ml of the second dilution to another 99 ml of water. I end up with a 10-2 X 10-2 X 10-2 = 10-6 dilution of the original bacteria culture. I can then plate .1 ml of the final dilution on growth medium. The goal is to dilute the culture so that, when plated, the number of bacterial colonies is discrete and each colony arises from one viable bacterial cell. We can use the number of viable cells in the undiluted culture by dividing apparent colony-forming units with the product of milliliters used and the dilution factor. For example, if 150 colony-forming units were counted on the plate that was streaked with .1 ml of the 10-6 dilution, there is about 150 / (.1 ml X 10-6) = 1.5 x 109 bacteria/ml in the original culture. This method is useful because I am using only a small portion of the original culture, and large volumes of solution are not required for many-fold dilutions.

The Petroff-Hausser Counting Chamber can also be used to validate cell counts. The cell suspension is vortexed and a drop is applied to the chamber with a Pasteur pipette. Etched squares on the surface of the chamber representing specific areas and volumes are then examined under high magnification. Count the number of bacterial cells per chamber cell and multiply to obtain the concentration of cells per milliliter.

The turbidimetric method indirectly determines the quantity of insoluble particles in a liquid by comparing light transmittance in reference to a standard solution. A spectrophotometer shines a specific wavelength of light at the sample. Insoluble particles suspended in the sample will absorb and the incidental light, decreasing the amount of light transmitted to the photocell. Optical density is the measure of the turbidity of a solution, and it increases as the concentration and size of the particles increase. For example, as the concentration of bacteria reaches about 107 cells per ml, the liquid medium will appear cloudy or turbid.

Aλ= log10(Io/I) = εbc

The absorption of light is described by the Beer-Lambert Law, where A is absorbance, Io is the intensity of light incident on the sample, and I is the intensity of light transmitted through the sample. Beer’s Law states the optical density is proportional to the concentration of the compound in the solution, c, and the light’s path length, b. Thus, the concentration of bacteria in a pure culture can be determined if the molar absorbtivity, ε, and the path length, c, are known. OD600 refers to the optical density of a sample when the incident light has a wavelength of 600 nanometers. 

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Evolution and G6PD Deficiency

Evolution takes place over thousands of years, when I asked about evolution, I was looking for an answer that had the same scope. I wasn't not talking about the past few decades, but the past few thousand years. So while some made very valid comments on botox and the nebulous cultural standards of beauty, the answer I liked best looked at the bigger picture and had some specific examples to support his thoughts.

M Fawlful made a good point about many genetic diseases becoming apparent later in life. These inherited diseases have no effect on the mating fitness of an individual. M Fawlful also stated that individuals heterozygous for the disease can actually be more fit and produce more affected progeny versus unaffected homozygotes. It's why some populations in Africa have sickle cell anemia; G6PD deficiency confers resistance to falciparum malaria (one of the biggest infectious killers in Africa).

The thing is, in developed countries, our environment is no longer selecting for any physical trait in particular. It is no longer putting a selective pressure against the unfit, because humankind has changed the environment to fit its needs.

We have made homes with air conditioning, built supermarkets and awesome hospitals, developed vaccines against polio and tetanus, for example. These allow everyone to live and thrive, regardless of their physical fitness or their potential skills as a hunter/provider.

A lot of you mentioned ugliness or perceived physical beauty was irrelevant when we're talking about sexual fitness, but I'm still not convinced. An individual's preference, influenced by upbringing or whatever, doesn't have as much influence on the evolutionary progress of a species. People are instinctively drawn to people who look a certain way. Having a symmetrical face is a sign of physical fitness. Having wide hips and large breasts is a good indication of a female's fertility, for example. That's what I mean by good-looks, but I digress.

Another good point made by many of you is that mutations are always popping up in our genomes, and these mutations lead to birth defects and weird traits like a long neck or whatever. Given that these mutations are the substrate on which evolution can take its course and new ones constantly pop up in every generation, ugliness and new genetic diseases can never be completely eradicated.

Ultimately, M Fawlful, I picked your comment out of the many great comments by Bellingham, Ghevrix, GMSoccerPicks, Inverse, Procras, Gareth Thomas, DS, Bersercules, Mekkor, Clueless Dolphin, Equalz, Electric Addict, Lars, Twist of Events, H., Maxe's Maze, Natural One, ason31, neversettleforsecond, Michael Westside, Randall A., Shaw, Timothy Bowen, Kid Shuffle, convictus, and last but not least, Bulletproof Zombie. To everyone who contributed to this very interesting discussion, thank you. To the people who have followed this website since July, thank you for your continued support. TheTruthAboutGenetics.com has 200 followers now.

Sources linked directly above.

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Question of the Day

Why hasn't evolution gotten rid of the ugly and genetically dysfunctional individuals? It's survival of the fittest, right? For the past hundred thousands of years, wouldn't ugliness and genetic diseases be slowly weeded out?

M Fawlful won the 8 GB SD card. Congratulations!

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