An 8-month-old, exclusively breastfed girl presented with a five-month history of vomiting with subsequent failure to thrive and lethargy. Family history was notable for the maternal history of gastroschisis. Mother had no dietary restrictions and had successfully breastfed multiple children for >12 months without issue. Initial evaluation was notable for macrocytic anemia. Subsequent serum B
12
levels were undetectable. Upon further questioning, the mother had significant bowel resection as an infant due to complications of gastroschisis. Maternal serum B
12
levels were also undetectable. The infant’s symptoms resolved with supplementation.
The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins.
Steady state myoplasmic ion concentration and slow ion transport across the sarcolemma can be quantitatively studied by means of ion sensing electrodes (ISE). ISE's readout represents the sum of the electrochemical potential of the ion of interest and the resting membrane potential (Vm). Vm is measured either by using an independent standard microelectrode or a double-barreled capillary. These approaches have known limitations. Ion transport and membrane potential are intricately connected, the resting potential may be unstable or be far from the desired value, ion transport can be electrically silent, and membrane currents may be carried by more than one ion. Thus, an electrophysiological method able to simultaneously measure the concentration of more than one ion, and to measure and control Vm and membrane current (Im) is desirable for physiological and pathophysiological studies. For skeletal fibers, a triple-barreled electrode has been used in the past allowing for current injection to modify Vm under current clamp conditions. Here we describe a novel four-microelectrode system composed of a two-microelectrode voltage-clamp amplifier (TEV-200A, Dagan) and a two channel high impedance electrometer (FD223a, WPI). This approach allows for potentiometric measurements of the concentration and transport of up to two ions in short murine skeletal muscle fibers subjected to either current-or voltage-clamp conditions. Ion translocation by primary and secondary active transport mechanisms, ion exchangers or passive ion diffusion can be studied. The system performance is exemplified by using Na, K and H selective electrodes made with commercially available ionophores and fibers enzymatically dissociated from the flexor digitorum brevis muscles.
The interaction sites between endoplasmic reticulum (ER) and mitochondria are signaling hubs in the cell implicated in calcium transfer, lipid metabolism, autophagy, and cell death among other functions. Transmission electron microscopy (TEM) is commonly used to visualize the geometry of these interfaces, but metrics used to describe and compare between conditions, as well as the techniques of measurement are not standardized. Here we describe a pair of scripts for ImageJ that allow for rapid, reproducible and flexible quantification of interface geometries. One script is focused on measuring the physical apposition of the membranes while the other considers the potential exposure of the outer mitochondrial membrane (OMM) to calcium released from the ER. In both cases, the user simply traces the OMM and nearby ER membranes; the script bins the interface distances and returns the length of the OMM that participates in an interface of a given gap width, as well as the total OMM and ER lengths. A 'score' based on the inverse-square of the distance is also generated as an attempt to make a single value representing 'interface-ness'. The additive nature of these measures allows cell-wise totals to be calculated for a given TEM section. While this approach standardizes the measurement technique for a number of parameters of ER-mitochondrial interfaces, it remains to be seen which parameters best correspond with physiological changes in different models. The ATPase inhibitory factor 1 (IF 1 ) is an ubiquitously expressed mitochondrial protein that blocks the reversal of the F 1 F o -ATPsynthase, preventing dissipation of cellular ATP and ischaemic damage. Many human cancers express high levels of IF 1 , which suppress cell death and enhance tumour cell invasion and chemoresistance. In this study, we assessed the effect of IF 1 over-expression on mitochondrial redox balance and apoptotic cristae remodelling. We found that IF 1 maintains ATP levels under apoptosis and reduces glutathione (GSH) loss and inactivation of peroxiredoxin 3 (Prx3). This correlates with inhibition of the metallopeptidase OMA1-mediated processing of the pro-fusion dynamin related protein optic atrophy 1 (OPA1), impeding cristae remodelling and apoptosis completion. IF 1 therefore has a pivotal antioxidant activity that hinders the OMA1/OPA1-dependent deconstruction of mitochondrial morphology and cellular demise. The data presented here highlight a dual regulatory activity of IF 1 on both mitochondrial bioenergetics and structure, resulting in increased proliferative capacity of tumour cells and significantly contributing to the molecular signalling of mitochondria in cancer.
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