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Introduction: Significant variations in Biological Variation (BV) estimates have been reported for urine markers. This study aimed to systematically review and critically appraise BV studies for albumin, creatinine, albumin-to-creatinine ratio (ACR), and other urine markers to perform a meta-analysis of eligible studies., Methods: Publications were identified through a systematic search and evaluated using the Biological Variation Data Critical Appraisal Checklist (BIVAC). BIVAC-compliant studies (grades A-C; A being fully compliant) conducted in healthy individuals were included in the meta-analysis, providing within-subject (CV I ) and between-subject (CV G ) BV estimates with 95% confidence intervals for various sample collection types., Results: Out of 37 studies evaluated, 16 were included (one grade A, three B, twelve C). No eligible publications were identified for meta-analysis of albumin and ACR. Limited data were available for first-morning urine specimens. A CV I between 15% and 30% was found for most measurands in 24-hour urine samples, while CV I estimates for random urine appeared higher., Conclusion: Published BV studies on urine markers utilized different sample collections and reporting units. Most were considered unfit for use or ineligible for meta-analysis. Given the critical role of urine albumin and ACR in chronic kidney disease risk assessment, there is a need for more BIVAC-compliant studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

There is a need for standards for generation and reporting of Biological Variation (BV) reference data. The absence of standards affects the quality and transportability of BV data, compromising important clinical applications. To address this issue, international expert groups under the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) have developed an online resource (https://tinyurl.com/bvmindmap) in the form of an interactive mind map that serves as a guideline for researchers planning, performing and reporting BV studies. The mind map addresses study design, data analysis, and reporting criteria, providing embedded links to relevant references and resources. It also incorporates a checklist approach, identifying a minimum data set (MDS) to enable the transportability of BV data and incorporates the Biological Variation Data Critical Appraisal Checklist (BIVAC) to assess study quality. The mind map is open to access and is disseminated through the EFLM BV Database website, promoting accessibility and compliance to a reporting standard, thereby providing a tool to be used to ensure data quality, consistency, and comparability of BV data. Thus, comparable to the STARD initiative for diagnostic accuracy studies, the mind map introduces a Standard for Reporting Biological Variation Data Studies (STARBIV), which can enhance the reporting quality of BV studies, foster user confidence, provide better decision support, and be used as a tool for critical appraisal. Ongoing refinement is expected to adapt to emerging methodologies, ensuring a positive trajectory toward improving the validity and applicability of BV data in clinical practice., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Analytical performance specifications (APS) are typically established through one of three models: (i) outcome studies, (ii) biological variation (BV), or (iii) state-of-the-art. Presently, The APS can, for most measurands that have a stable concentration, be based on BV. BV based APS, defined for imprecision, bias, total allowable error and allowable measurement uncertainty, are applied to many different processes in the laboratory. When calculating APS, it is important to consider the different APS formulae, for what setting they are to be applied and if they are suitable for the intended purpose. In this opinion paper, we elucidate the background, limitations, strengths, and potential intended applications of the different BV based APS formulas. When using BV data to set APS, it is important to consider that all formulae are contingent on accurate and relevant BV estimates. During the last decade, efficient procedures have been established to obtain reliable BV estimates that are presented in the EFLM biological variation database. The database publishes detailed BV data for numerous measurands, global BV estimates derived from meta-analysis of quality-assured studies of similar study design and automatic calculation of BV based APS., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Allowable total error (ATE) are performance specification limits predefined for a variety of laboratory analytes. These limits define the maximum amount of error that is allowed for an assay when judging acceptability of a new assay during method verification/validation, evaluating patient or instrument comparison data, or in designing a quality control strategy. There are several widely available resources and models that can serve as a guide in selecting ATE. They may be based on legal requirements or set by providers of proficiency testing (PT) and external quality assessment schemes (EQAS). ATE can be also determined by professional expert groups or be based on biological variation of an analyte. Because there are several resources to choose from, there have been several attempts in reaching consensus on which ATE resource should be given preference. This chapter reviews several of these resources in more detail and discusses the difference between allowable total error (ATE) and observed total analytical error (TAE)., (Copyright © 2024. Published by Elsevier Inc.)

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.

Over the past two decades, vitamin D level measurements have become some of the most frequently ordered tests in the laboratory. This increase is due to a growing awareness of widespread vitamin D deficiency and scientific data suggesting the beneficial effects of vitamin D in various diseases. A literature search was carried out in PubMed for cases reporting vitamin D intoxication and overdose. Thirteen articles were included in this review. Intoxication was severe in the reported cases. Patients presented with serum vitamin D concentrations ranging between 150 and 1220 ng/mL and serum calcium concentrations between 11.1 and 23.1 mg/dL. Most of the reported patients showed symptoms of vitamin D toxicity such as vomiting, dehydration, pain, and loss of appetite. The underlying causes included manufacturing errors, overdosing by patients or prescribers, and combinations of these factors. Our literature search highlights the fact that even though vitamin D intoxication is rare, it does occur and therefore patients and prescribers should be more cognizant of the potential dangers of vitamin D overdose.

Given that dysregulation of mechanics contributes to diseases ranging from cancer metastasis to lung disease, it is important to develop methods for screening the efficacy of drugs that target cellular forces. Here, nanoparticle-based tension sensors are used to quantify the mechanical response of individual cells upon drug treatment. As a proof-of-concept, the activity of bronchodilators is tested on human airway smooth muscle cells derived from seven donors, four of which are asthmatic. It is revealed that airway smooth muscle cells isolated from asthmatic donors exhibit greater traction forces compared to the control donors. Additionally, the mechanical signal is abolished using myosin inhibitors or further enhanced in the presence of inflammatory inducers, such as nicotine. Using the signal generated by the probes, single-cell dose-response measurements are performed to determine the "mechano" effective concentration (mechano-EC 50 ) of albuterol, a bronchodilator, which reduces integrin forces by 50%. Mechano-EC 50 values for each donor present discrete readings that are differentially enhanced as a function of nicotine treatment. Importantly, donor mechano-EC 50 values varied by orders of magnitude, suggesting significant variability in their sensitivity to nicotine and albuterol treatment. To the best of the authors' knowledge, this is the first study harnessing a piconewton tension sensor platform for mechanopharmacology., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In early 2000's vitamin-D deficiency was shown to be prevalent in several countries including the United States (US). Studies exploring the role of vitamin-D metabolism in diverse disease pathways generated an increased demand for vitamin-D supplementation and an immense public interest in measurement of vitamin-D metabolite levels. In this report, we review the role of vitamin-D metabolism in disease processes, clinical utility of measuring vitamin-D metabolites including 25-hydroxyvitamin-D (25(OH)D), 1,25-dihydroxyvitamin-D and 24,25-dihydroxyvitamin-D and discuss vitamin-D assay methodologies including immunoassays and liquid chromatography mass spectrometry (LC-MS/MS) assays. We also provide examples of vitamin-D toxicity and insight into the trends in serum 25(OH)D levels in the US population based on 10 years of data from on serum 25(OH)D values from ~5,000,000 patients who were tested at the Mayo Medical Laboratories between February 2007-February 2017.

Single-molecule force spectroscopy techniques are powerful tools for investigating the mechanical unfolding of biomolecules. However, they are limited in throughput and require dedicated instrumentation. Here, we report a force-generating particle that can unfold target molecules on-demand. The particle consists of a plasmonic nanorod core encapsulated with a thermoresponsive polymer shell. Optical heating of the nanorod leads to rapid collapse of the polymer, thus transducing light into mechanical work to unfold target molecules. The illumination tunes the duration and degree of particle collapse, thus controlling the lifetime and magnitude of applied forces. Single-molecule fluorescence imaging showed reproducible mechanical unfolding of DNA hairpins. We also demonstrate the triggering of 50 different particles in <1 min, exceeding the speed of conventional atomic force microscopy. The polymer force clamp represents a facile and bottom-up approach to force manipulation.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.

Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration, and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T- or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction. Upon ligation, these receptor-ligand bonds transmit piconewton (pN) mechanical forces that are generated, in part, by the cytoskeleton. Importantly, these forces expose cryptic sites within mechanosensitive proteins and modulate the binding kinetics (on/off rate) of receptor-ligand complexes to further fine-tune mechanotransduction and the corresponding cell behavior. Over the past three decades, two categories of methods have been developed to measure cell receptor forces. The first class is traction force microscopy (TFM) and micropost array detectors (mPADs). In these methods, cells are cultured on elastic polymers or microstructures that deform under mechanical forces. The second category of techniques is single molecule force spectroscopy (SMFS) including atomic force microscopy (AFM), optical or magnetic tweezers, and biomembrane force probe (BFP). In SMFS, the experimenter applies external forces to probe the mechanics of individual cells or single receptor-ligand complexes, serially, one bond at a time. Although these techniques are powerful, the limited throughput of SMFS and the nN force sensitivity of TFM have hindered further elucidation of the molecular mechanisms of mechanotransduction. In this Account, we introduce the recent advent of molecular tension fluorescence microscopy (MTFM) as an emerging tool for molecular imaging of receptor mechanics in living cells. MTFM probes are composed of an extendable linker, such as polymer, oligonucleotide, or protein, and flanked by a fluorophore and quencher. By measuring the fluorescence emission of immobilized MTFM probes, one can infer the extension of the linker and the externally applied force. Thus, MTFM combines aspects of TFM and SMFS to optically report receptor forces across the entire cell surface with pN sensitivity. Specifically, we provide an in-depth review of MTFM probe design, which includes the extendable "spring", spectroscopic ruler, surface immobilization chemistry, and ligand design strategies. We also demonstrate the strengths and weaknesses of different versions of MTFM probes by discussing case studies involving the pN forces involved in epidermal growth factor receptor, integrin, and T-cell receptor signaling pathways. Lastly, we present a brief future outlook, primarily from a chemists' perspective, on the challenges and opportunities for the design of next generation MTFM probes.

Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks (p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks (p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise regardless of diet. Plin5 was increased with HFD and unaffected by exercise; the respiratory exchange ratio was 15% lower in the 18-week HFD group compared with CTL (p < 0.001) and 10% lower in 18 weeks HFD-E compared with CTL-E (p < 0.001). Increased Ucp1 and Pgc1a in exercised muscle of running mice suggests that a beige/brown fat phenotype develops, which differs from the fat phenotype that induces insulin resistance in high fat feeding. This suggests that increased muscle lipid may develop a "brown" phenotype in the setting of endurance exercise training, a shift that is further promoted by HFD.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Mechanics play a fundamental role in cell biology, but detecting piconewton (pN) forces is challenging because of a lack of accessible and high throughput assays. A mechanically induced catalytic amplification reaction (MCR) for readout of receptor-mediated forces in cells is described. Mechanically labile DNA duplexes presenting ligands are surface immobilized such that specific receptor forces denature the duplex and thus expose a blocked primer. Amplification of primers is achieved using an isothermal polymerization reaction and quantified by fluorescence readout. As a proof of concept, the assay was used to test the activity of a mechanomodulatory drug and integrin adhesion receptor antibodies. To the best of our knowledge, this is the first example of a catalytic reaction triggered in response to molecular piconewton forces. The MCR may transform the field of mechanobiology by providing a new facile tool to detect receptor specific mechanics with the convenience of the polymerase chain reaction (PCR)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mechanical forces between cells and their extracellular matrix (ECM) are mediated by dozens of different receptors. These biophysical interactions play fundamental roles in processes ranging from cellular development to tumor progression. However, mapping the spatial and temporal dynamics of tension among various receptor-ligand pairs remains a significant challenge. To address this issue, we have developed a synthetic strategy to generate modular tension probes combining the native chemical ligation (NCL) reaction with solid phase peptide synthesis (SPPS). In principle, this approach accommodates virtually any peptide or expressed protein amenable to NCL. We generated a small library of tension probes displaying different ligands, flexible linkers, and fluorescent reporters, enabling the mapping of integrin and cadherin tension, and demonstrating the first example of long-term (∼3 days) molecular tension imaging. This approach provides a toolset to better understand mechanotransduction events fundamental to cell biology.

Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30-40 pN. The addition of a disulfide bridge within I27 "clamps" the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S-S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S-S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported.

The contribution of marrow adipose tissue (MAT) to skeletal fragility is poorly understood. Peroxisome proliferator-activated receptor (PPAR)γ agonists, associated with increased fractures in diabetic patients, increase MAT. Here, we asked whether exercise could limit the MAT accrual and increase bone formation in the setting of PPARγ agonist treatment. Eight-week-old female C57BL/6 mice were treated with 20-mg/kg · d rosiglitazone (Rosi) and compared with control (CTL) animals. Exercise groups ran 12 km/d when provided access to running wheels (CTL exercise [CTL-E], Rosi-E). After 6 weeks, femoral MAT (volume of lipid binder osmium) and tibial bone morphology were assessed by microcomputer tomography. Rosi was associated with 40% higher femur MAT volume compared with CTL (P < .0001). Exercise suppressed MAT volume by half in CTL-E mice compared with CTL (P < .01) and 19% in Rosi-E compared with Rosi (P < .0001). Rosi treatment increased fat markers perilipin and fatty acid synthase mRNA by 4-fold (P < .01). Exercise was associated with increased uncoupling protein 1 mRNA expression in both CTL-E and Rosi-E groups (P < .05), suggestive of increased brown fat. Rosi increased cortical porosity (P < .0001) but did not significantly impact trabecular or cortical bone quantity. Importantly, exercise induction of trabecular bone volume was not prevented by Rosi (CTL-E 21% > CTL, P < .05; Rosi-E 26% > Rosi, P < .01). In summary, despite the Rosi induction of MAT extending well into the femoral diaphysis, exercise was able to significantly suppress MAT volume and induce bone formation. Our results suggest that the impact of PPARγ agonists on bone and marrow health can be partially mitigated by exercise.

Herein we aimed to understand how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We combined molecular tension fluorescence microscopy with block copolymer micelle nanolithography to fabricate substrates with arrays of precisely spaced probes that can generate a 10-fold fluorescence response to pN-forces. We found that the mechanism of sensing ligand spacing is force-mediated. This strategy is broadly applicable to investigating receptor clustering and its role in mechanotransduction pathways.

Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic insufficiency, remains poorly understood and difficult to quantify. We tested the response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis, and compared it to measures of bone quantity. We hypothesized that HFD would increase MAT and diminish bone quantity, while exercise would slow MAT acquisition and promote bone formation. Eight week-old female C57BL/6 mice were fed a regular (RD) or HFD, and exercise groups were provided voluntary access to running wheels (RD-E, HFD-E). Femoral MAT was assessed by μCT (lipid binder osmium) using a semi-automated approach employing rigid co-alignment, regional bone masks and was normalized for total femoral volume (TV) of the bone compartment. MAT was 2.6-fold higher in HFD relative to RD mice. Exercise suppressed MAT in RD-E mice by more than half compared with RD. Running similarly inhibited MAT acquisition in HFD mice. Exercise significantly increased bone quantity in both diet groups. Thus, HFD caused significant accumulation of MAT; importantly running exercise limited MAT acquisition while promoting bone formation during both diets. That MAT is exquisitely responsive to diet and exercise, and its regulation by exercise appears to be inversely proportional to effects on exercise induced bone formation, is relevant for an aging and sedentary population., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Exercise prevents marrow mesenchymal stem cell (MSC) adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPARγ expression and adipogenesis in MSC. We considered whether C/EBPβ might be mechanoresponsive as it is upstream to PPARγ, and also is known to upregulate endoplasmic reticulum (ER) stress. MSC (C3H10T1/2 pluripotent cells as well as mouse marrow-derived MSC) were cultured in adipogenic media and a daily mechanical strain regimen was applied. We demonstrate herein that mechanical strain represses C/EBPβ mRNA (0.6-fold ±0.07, p<0.05) and protein (0.4-fold ±0.1, p<0.01) in MSC. SiRNA silencing of β-catenin prevented mechanical repression of C/EBPβ. C/EBPβ overexpression did not override strain's inhibition of adipogenesis, which suggests that mechanical control of C/EBPβ is not the primary site at which adipogenesis is regulated. Mechanical inhibition of C/EBPβ, however, might be critical for further processes that regulate MSC health. Indeed, overexpression of C/EBPβ in MSC induced ER stress evidenced by a dose-dependent increase in the pro-apoptotic CHOP (protein 4-fold ±0.5, p<0.05) and a threshold reduction in the chaperone BiP (protein 0.6-fold ±0.1, p = 0.2; mRNA 0.3-fold ±0.1, p<0.01). ChIP-seq demonstrated a significant association between C/EBPβ and both CHOP and BiP genes. The strain regimen, in addition to decreasing C/EBPβ mRNA (0.5-fold ±0.09, p<0.05), expanded ER capacity as measured by an increase in BiP mRNA (2-fold ±0.2, p<0.05) and protein. Finally, ER stress induced by tunicamycin was ameliorated by mechanical strain as demonstrated by decreased C/EBPβ, increased BiP and decreased CHOP protein expression. Thus, C/EBPβ is a mechanically responsive transcription factor and its repression should counter increases in marrow fat as well as improve skeletal resistance to ER stress.

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.