Membrane stretch as the mechanism of activation of PIEZO1 ion channels in chondrocytes.

Osteoarthritis is a chronic disease that can be initiated by altered joint loading or injury of the cartilage. The mechanically sensitive PIEZO ion channels have been shown to transduce injurious levels of biomechanical strain in articular chondrocytes and mediate cell death. However, the mechanisms of channel gating in response to high cellular deformation and the strain thresholds for activating PIEZO channels remain unclear. We coupled studies of single-cell compression using atomic force microscopy (AFM) with finite element modeling (FEM) to identify the biophysical mechanisms of PIEZO-mediated calcium (Ca2+) signaling in chondrocytes. We showed that PIEZO1 and PIEZO2 are needed for initiating Ca2+ signaling at moderately high levels of cellular deformation, but at the highest strains, PIEZO1 functions independently of PIEZO2. Biophysical factors that increase apparent chondrocyte membrane tension, including hypoosmotic prestrain, high compression magnitudes, and low deformation rates, also increased PIEZO1-driven Ca2+ signaling. Combined AFM/FEM studies showed that 50% of chondrocytes exhibit Ca2+ signaling at 80 to 85% nominal cell compression, corresponding to a threshold of apparent membrane finite principal strain of E = 1.31, which represents a membrane stretch ratio (λ) of 1.9. Both intracellular and extracellular Ca2+ are necessary for the PIEZO1-mediated Ca2+ signaling response to compression. Our results suggest that PIEZO1-induced signaling drives chondrocyte mechanical injury due to high membrane tension, and this threshold can be altered by factors that influence membrane prestress, such as cartilage hypoosmolarity, secondary to proteoglycan loss. These findings suggest that modulating PIEZO1 activation or downstream signaling may offer avenues for the prevention or treatment of osteoarthritis.

The role of PIEZO ion channels in the musculoskeletal system.

PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. Although many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.

Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity.

Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.

Comparison of the effects of virtual training by serious game and lecture on operating room novices' knowledge and performance about surgical instruments setup: a multi-center, two-arm study.

BACKGROUND: Game-based training is increasingly implemented in different nursing fields, as it allows students to learn experientially, with the flexibility to regulate their training based on their personal progresses and abilities. This study aimed to compare the effects of virtual training by the "Playing with Surgical Instruments (PlaSurIn)" game and the lecture on the surgical instruments setup knowledge and performance of Operating Room (OR) novices. METHODS: This study was conducted on 51 s-semester undergraduate OR technology students taking the course "An Introduction to Surgical Instruments and Equipment." An additional virtual training session was held via a learning management system using two different methods. The students of the Game Training Group (GTG, n = 27) played individually with the "PlaSurIn" game during a week, while the students of the Lecture Training Group (LTG, n = 24) received the lecture-based training during a week. To measure knowledge, all the students participated in a theoretical test with 10 multiple-choice questions before and immediately after the training. They also participated in an Objective Structured Clinical Examination (OSCE) after the training, and their performance was evaluated by the remained time for setup completion and the scores, errors, and bonuses. RESULTS: The mean score of the theoretical test was significantly higher in the GTG than in the LTG after the training (p = 0.040). Additionally, the GTG participants had higher scores (p = 0.016), fewer errors (p = 0.001), and higher bonuses (p = 0.011) compared to the LTG ones. The remained time for setup completion was also significantly longer in the GTG than in the LTG (p < 0.001). CONCLUSION: Virtual training by "PlaSurIn" was superior to the lecture-based method for the enhancement of surgical instruments setup knowledge and performance amongst OR novices.

Iris stromal cell nuclei deform to more elongated shapes during pharmacologically-induced miosis and mydriasis.

Nuclear shape alteration in ocular tissues, which can be used as a metric for overall cell deformation, may also lead to changes in gene expression and protein synthesis that could affect the biomechanics of the tissue extracellular matrix. The biomechanics of iris tissue is of particular interest in the study of primary angle-closure glaucoma. As the first step towards understanding the mutual role of the biomechanics and deformation of the iris on the activity of its constituent stromal cells, we conducted an ex-vivo study in freshly excised porcine eyes. Iris deformation was achieved by activating the constituent smooth muscles of the iris. Pupillary responses were initiated by inducing miosis and mydriasis, and the irides were placed in a fixative, bisected, and sliced into thin sections in a nasal and temporal horizontal orientation. The tissue sections were stained with DAPI for nucleus, and z-stacks were acquired using confocal microscopy. Images were analyzed to determine the nuclear aspect ratio (NAR) using both three-dimensional (3D) reconstructions of the nuclear surfaces as well as projections of the same 3D reconstruction into flat two-dimensional (2D) shapes. We observed that regardless of the calculation method (i.e., one that employed 3D surface reconstructions versus one that employed 2D projected images) the NAR increased in both the miosis group and the mydriasis group. Three-dimensional quantifications showed that NAR increased from 2.52 ± 0.96 in control group to 2.80 ± 0.81 and 2.74 ± 0.94 in the mydriasis and miosis groups, respectively. Notwithstanding the relative convenience in calculating the NAR using the 2D projected images, the 3D reconstructions were found to generate more physiologically realistic values and, thus, can be used in the development of future computational models to study primary angle-closure glaucoma. Since the iris undergoes large deformations in response to ambient light, this study suggests that the iris stromal cells are subjected to a biomechanically active micro-environment during their in-vivo physiological function.

Response of broiler chickens reared at high density to dietary supplementation with licorice extract and probiotic.

This study evaluated the effects of licorice extract and probiotic on performance, excreta characteristics and welfare-related parameters of broiler chickens reared in high stocking density. Three hundred and thirty-six 1-day old broiler chicks (Ross 308) were used in a completely randomized design with five treatments and four replicates for 42 days. Treatments were as follows: (a) positive control (PC, 12 chicks/m2 ); (b) negative control (NC, 18 chicks/m2 ); (c) NC + 500 ppm licorice extract (LE); (d) NC + 200 ppm probiotic (P); (e) NC + ppm LE + ppm P. Body weight gain (BWG) was affected negatively by increased density in the growing period (p < 0.05). At high density, BWG was increased in the growing and whole period by adding LE and P separately and in combination (p < 0.01). Feed conversion ratio (FCR) was not affected by stocking density. Excreta weight (g/bird) and dry matter (DM, %) were reduced by increasing density (p < 0.01). Use of feed additives at high stocking density had no significant effect on excreta weight, but DM content of excreta was higher in NC + LE + P than NC. Gait problems and footpad and hock burns were increased by increased stocking density, but were not altered by feed additives. In conclusion, licorice extract and probiotic improved BWG of broilers reared at high stocking density; however, these birds had some disorders in footpad, hock and walking ability.

"Playing with Surgical Instruments (PlaSurIn)" game to train operating room novices how to set up basic surgical instruments: A validation study.

BACKGROUND: Game-based training has been considered as an alternative modality to traditional training in different perioperative nursing fields. OBJECTIVES: To describe the adaptation and validation process of "Playing with Tweezers", a Portuguese game developed for novices to set up basic surgical instruments on the Mayo stand or a back table. DESIGN: A validation study with three phases of translation, reconciliation, and evaluation (face, content, and construct validity). SETTINGS: Several medical universities in Iran. PARTICIPANTS: Twelve students in a pilot translation test, 18 experts in the reconciliation phase, 20 experts in the face and content validity stages, and 120 students (72 novices, 26 intermediates, and 22 experts) in the construct validity stage. METHODS: Following "forward-backward" translation from Portuguese to English, the English version of the game was appraised in the reconciliation phase using a 57-item questionnaire. To test face and content validity of the final version of the game, a 30-item questionnaire addressing different aspects of the game was completed. The students' game performance (remained time for game completion, obtained score, and error) was compared to assess the construct validity. RESULTS: Minor differences were detected and resolved during the translation process. The English version of the game was reconciled in two sequential steps, and the final game called "Playing with Surgical Instruments (PlaSurIn)" was developed. All the items regarding the face validity received 80-100% of positive responses. Moreover, regarding the content validity, all of the evaluated items obtained a content validity index of 0.90-1.0. Compared to the novices, the experts and intermediates received higher scores (p < 0.001 in two cases) and fewer errors (p < 0.001, p = 0.007). The remained time for game completion was significantly longer for experts than the novices (p = 0.011). CONCLUSIONS: The "PlaSurIn", as a virtual training strategy, can prepare novices to set up basic surgical instruments.

Theoretical Assessment of the Risk of Ocular Hypotony in Patients With Intravitreal Gas Bubbles Who Travel Through Subsea Tunnels.

PURPOSE: This study was conducted to investigate changes in intraocular pressure (IOP) in the presence of intravitreal gas bubbles in individuals who travel through subsea tunnels. METHODS: Using a mathematical model, we simulated alterations in ocular globe shape, aqueous humor flow, volume of intravitreal gas bubbles, and IOP due to elevation changes during travel through subsea tunnels. We simulated five tunnels with different features as case studies. The role of key modeling parameters was further evaluated in a parametric study. RESULTS: In three out of the five simulated tunnels (i.e., Seikan Tunnel, Bomlafjord Tunnel, and the Atlantic Ocean Tunnel), the patients were potentially at risk at lower portions of the tunnels since the IOP dropped to values less than 5 mm Hg, the clinical threshold for ocular hypotony. During ascent, the IOP increased to the normal value of 15 mm Hg and in some cases to higher values (e.g., a peak value of 22 mm Hg in Seikan Tunnel). CONCLUSIONS: Our model predicted that in the presence of intravitreal gas bubbles, the IOP could drop to extremely low values when patients descend to lower elevations in some tunnels. Such low IOP values could cause bleeding and/or retinal detachment. Since many factors (e.g., tunnel specifications and/or patient-specific characteristics) could affect the IOP during subsea travel, caution (beyond avoiding airplane flights) should be taken in advising patients about travel restrictions following intravitreal gas injections. TRANSLATIONAL RELEVANCE: Our findings highlight the potential risk for hypotony in the presence of intravitreal gas bubbles during subsea travels.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-ß1.

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-ß1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.