Osteocytes sense loading in bone, but their mechanosensation mechanisms remain poorly understood. Plasma membrane disruptions (PMD) develop with loading under physiological conditions in many cell types (e.g., myocytes, endothelial cells). These PMD foster molecular flux across cell membranes that promotes tissue adaptation, but this mechanosensation mechanism had not been explored in osteocytes. Our goal was to investigate whether PMD occur and initiate consequent mechanotransduction in osteocytes during physiological loading. We found that osteocytes experience PMD during in vitro (fluid flow) and in vivo (treadmill exercise) mechanical loading, in proportion to the level of stress experienced. In fluid flow studies, osteocyte PMD preferentially formed with rapid as compared to gradual application of loading. In treadmill studies, osteocyte PMD increased with loading in weight bearing locations (tibia), but this trend was not seen in non-weight bearing locations (skull). PMD initiated osteocyte mechanotransduction including calcium signaling and expression of c-fos, and repair rates of these PMD could be enhanced or inhibited pharmacologically to alter downstream mechanotransduction and osteocyte survival. PMD may represent a novel mechanosensation pathway in bone and a target for modifying skeletal adaptation signaling in osteocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:653-662, 2018.
The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena , carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena , loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas , mammalian CCRK and DYF-18 of C . elegans , in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.
Osteocytes experience plasma membrane disruptions (PMD) that initiate mechanotransduction both in vitro and in vivo in response to mechanical loading, suggesting that osteocytes use PMD to sense and adapt to mechanical stimuli. PMD repair is crucial for cell survival; antioxidants (e.g., alpha-tocopherol, also known as Vitamin E) promote repair while reactive oxygen species (ROS), which can accumulate during exercise, inhibit repair. The goal of this study was to determine whether depleting Vitamin E in the diet would impact osteocyte survival and bone adaptation with loading. Male CD-1 mice (3 wk old) were fed either a regular diet (RD) or Vitamin E-deficient diet (VEDD) for up to 11 weeks. Mice from each dietary group either served as sedentary controls with normal cage activity, or were subjected to treadmill exercise (one bout of exercise or daily exercise for 5 weeks). VEDD-fed mice showed more PMD-affected osteocytes (+50%) after a single exercise bout suggesting impaired PMD repair following Vitamin E deprivation. After 5 weeks of daily exercise, VEDD mice failed to show an exercise-induced increase in osteocyte PMD formation, and showed signs of increased osteocytic oxidative stress and impaired osteocyte survival. Surprisingly, exercise-induced increases in cortical bone formation rate were only significant for VEDD-fed mice. This result may be consistent with previous studies in skeletal muscle, where myocyte PMD repair failure (e.g., with muscular dystrophy) initially triggers hypertrophy but later leads to widespread degeneration. In vitro, mechanically wounded MLO-Y4 cells displayed increased post-wounding necrosis (+40 fold) in the presence of H 2 O 2 , which could be prevented by Vitamin E pre-treatment. Taken together, our data support the idea that antioxidant-influenced osteocyte membrane repair is a vital aspect of bone mechanosensation in the osteocytic control of PMD-driven bone adaptation.
22The length of cilia is controlled by a poorly understood mechanism that involves 23 members of the conserved RCK kinase group, and among them, the LF4/MOK 24 kinases. In Tetrahymena, a loss of an LF4/MOK ortholog, LF4A, lengthened the 25 locomotory cilia, but also reduced their total number per cell. Without LF4A, cilia 26 assembled faster and showed signs of increased intraflagellar transport (IFT). 27Consistently, overproduced LF4A shortened cilia and downregulated the IFT. GFP-28 tagged LF4A, expressed in the native locus and imaged by total internal reflection 29 microscopy, was enriched at the basal bodies and distributed along the shafts of 30 cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or 31 moved by IFT. A forward genetic screen identified a CDK-related kinase, CDKR1, 32 whose loss-of-function suppressed the shortening of cilia caused by overexpression 33 of LF4A, by reducing its kinase activity. A loss of CDKR1 alone lengthened both the 34 locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: 35 LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the 36 cyclin-binding motif and acting upstream of RCKs. We propose that the total 37 LF4/MOK activity per cilium is dependent on both its activation by an upstream CDK-38 related kinase and cilium length. Previous studies showed that the rate of assembly 39 is high in growing cilia and decreases as cilia elongate to achieve the steady-state 40 length. We propose that in a longer cilium, the IFT components, which travel from 41 the base to the tip, are subjected to a higher dose of inhibition by the uniformly 42 3 distributed LF4/MOK. Thus, in a feedback loop, LF4/MOK may translate cilium length 43 into proportional inhibition of IFT, to balance the rates of assembly and disassembly 44 at steady-state. 45 46Author summary 47 Cilia are conserved organelles that generate motility and mediate vital sensory 48 functions, including olfaction and vision. Cilia that are either too short or too long fail 49 to generate proper forces or responses to extracellular signals. Several cilia-based 50 diseases (ciliopathies) are associated with defects in cilia length. Here we use the 51 multiciliated model protist Tetrahymena, to study a conserved protein kinase whose 52 activity shortens cilia, LF4/MOK. We find that cells lacking an LF4/MOK kinase of 53Tetrahymena, LF4A, have excessively long, but also fewer cilia. We show that LF4A 54 decreases the intraflagellar transport, a motility that shuttles ciliary precursors from 55 the cilium base to the tip. Live imaging revealed that LF4A is distributed along cilium 56 length and remains mostly immobile, likely due to its anchoring to ciliary 57 microtubules. We proposed that in longer cilia, the intraflagellar transport machinery 58 is exposed to a higher dose of inhibition by LF4A, which could decrease the rate of 59 cilium assembly, to balance the rate of cilium disassembly in mature cilia that 60 maintain stable length. 61 62 63T...
Dexmedetomidine is an alpha-2 agonist sedative and analgesic used in anesthesia practice, and it has become more prevalent in the critically ill patients requiring short-term mechanical ventilation. While dexmedetomidine is known to have minimal effects on respiratory drive, it has been well-documented to cause bradycardia and hypotension, especially in patients with existing comorbidities. We present a patient without cardiovascular comorbidities who was in the surgical ICU under dexmedetomidine sedation. The patient went into asystole cardiac arrest after vagal stimulation. Return of spontaneous circulation was achieved using ACLS protocol. We offer a review of reported cases and make recommendations on the management of similar situations that may arise given the increasing use of dexmedetomidine.
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