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Forces are produced, supported and transmitted by molecular elements such as cytoskeletal and adhesion components. FRET can be used to measure the amplitude of tension that these molecules experience in tissues. A FRET tension sensor module consists of two fluorophores: a donor and an acceptor ( Miyawaki, 2011 ; Gayrard and Borghi, 2015 ; Fig.3 Aa). FRET efficiency ( E ) decreases sharply with the distance R between fluorophores according to the equation E=R 0 6 /( R 6 + R 0 6 ), where R 0 is the distance at which FRET efficiency is 50% ( Fig.3 Ab). A spring of known stiffness, such as a defined polypeptide sequence, is genetically inserted between donor and acceptor ( Meng et al., 2008 ; loose fit shirt dress Pink amp; Purple Philosophy di Lorenzo Serafini hZlKXdH
). A force pulling on the molecule increases the spring length, thus reducing FRET efficiency. The FRET efficiency-force relationship is calibrated in vitro ; typically, it probes from one to several pN ( Grashoff et al., 2010 ; Fig.3 Ac). This technique relies on the assumption that molecular tension is the only cause of FRET change, which has to be tested using tensionless control constructs, e.g. lacking the N- or C-termini of the encoded proteins, under the same perturbations as their tension-sensing counterpart. In principle, this allows non-tension contributions to FRET changes, regardless of their origin, to be corrected for.

Fig. 3.

Visual sensors. (A) FRET. (Aa) FRET tension sensor module consists of two fluorophores, a donor (blue) and an acceptor (green), linked with a spring of known stiffness, the length of which increases with pulling forces (arrows) acting on it. (Ab) Energy transfer efficiency (light gray arrows in Aa) sharply decreases with the distance between fluorophores. (Ac) The FRET efficiency-force relationship is experimentally calibrated . Red line indicates a fitting curve. (Ad) FRET tension sensor module inserted into DE-cadherin molecules can measure their tension. FRET efficiency (color bar) in border cells of ovary is shown. (B) Liquid drop. (Ba) A liquid drop made of fluorocarbon oil (light orange) is coated by surfactant molecules, fluorescent molecules and adhesion molecules. The surface tension () of an isolated drop is measured in solution (left panel). When the liquid drop is inserted between cells in a tissue (right panel), it is deformed by cellular forces (arrows). Image analysis determines the local mean curvature of the droplet surface (), from which the anisotropic component of the normal stresses on the droplet (σ) can be determined according to the equation σ 2γ. (Bb) Liquid drops (red) injected between tooth mesenchymal cells (green). Scale bar: 20 µm. The values of the anisotropic stresses (color bar) are mapped onto the three-dimensional shape of the liquid drop (enlarged in inset). Adapted with permission from: (Ad) Cai et al. (2014 ) and (Ba, Bb) Campàs et al. (2014 ).

A major advantage of the method is that it could be applicable to any protein of interest as long as the tagged protein (containing the tension sensor) retains the localization and functions of the native protein. In addition, the method is, in principle, compatible with imaging of other proteins and/or structures, other force measurements, or any mechanical manipulation performed on a microscope stage. Within a very short time period (the time for image acquisition, typically around 100 ms), a map of molecular tensions (for the tagged molecule) in a tissue can be obtained in vivo : each pixel intensity results from the sum of FRET efficiencies of the molecules within this pixel. In cell culture, FRET has also been used to measure uniaxial compression ( Paszek et al., 2014 ). More generally, fluorescent materials exhibit a variety of properties that could be dependent on other mechanical quantities, such as intracellular pressure ( Gomez-Martinez et al., 2013 ; Watanabe et al., 2013 ), and thus could be used to develop new force probes.

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Hope, I had a very similar experience and felt like I had more energy and muscle strength after starting B12 shots. I had to increase my intake of Potassium to about 2400 mg a day as my body was using more potassium because it was no longer starved for B12. I still have problems but the B12 shots helped a lot.


Gregory says

Dear Hope, I am not at all surprised at the difference between the shots and the sublingual. The sublingual is not really a very good mode of delivery, and there is no conclusive evidence that shows it is better than high dose tablets. It is based upon an incorrect premise that there are blood vessels near the surface of the underside of the tongue and so the material should go into them. Well that is the problem, it doesn’t, and I don’t know of any study that has shown it does. In the intestine you have a massive area for uptake of vitamin B12, much, much bigger than under the tongue. Best estimates for 1 mg sublingual tablet, 1 mg sublingual spray or 1 mg oral tablet is that up to 10 ug gets in. If you are deficient, you are down somewhere between 10-50 mg, or around 1,000 to 50,000 times more than you can aborb by either of the 3 methods. That is why the shots are so much better. It is also why we are working on a transdermal oil that gets around the same amount as the shots.


I started to get excellent results from sublingual B12 when I began to put the tablet between my cheek and gum and hold it there for 1-2 hours. It is absorbed slowly, chewing it up doesn’t work. If you continue with B12 shots, make sure you are getting the active form, methylcobalamin, not the cheap imitation stuff. I also use adenosylcobalamin, the other active form of b12, marketed as dibencozide, with great results.


sue says

HI, everyone. Just wanted to say how we underestimate Vitamin B12. My son has ASpergers syndrome. Last winter, out of the blue, he had Total Bell’s Palsy. It took him 6 months for the cheek to move, and 12 months for the nerve totally recuperate, even though his left side of face is still drooping. Everyone(including my son’s father, who is a doctor) were saying that Bell’s palsy comes only once. Well, guess what? 4 weeks ago, my son ‘s right side of the face was affected with nasty Bell’s palsy again!! His neurologist gave him anti viral and steroid. When my son came back(he is 21 years old, he was visiting his father during 2nd Bell’s occurrence) , I read so much about B12 methylcobalamine. I immediately gave him injections every other day. BELIEVE ME, AFTER 1ST INJECTION, HIS LIP STARTED MOVING. AFTER ONE WEEK, HIS FACE HAS COMPLETELY HEALED, MOVING, SMILING!!!!! MY SON’S FATHER, WHO IS A DOCTOR, SAID THAT THIS WAS JUST A COINCIDENCE . i give my son injections 1mg every other day. Hi stopped having twitches(he always had them), he is smiling and moving his lips.


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