The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

DrugSynthMC: An Atom-Based Generation of Drug-like Molecules with Monte Carlo Search.

A growing number of deep learning (DL) methodologies have recently been developed to design novel compounds and expand the chemical space within virtual libraries. Most of these neural network approaches design molecules to specifically bind a target based on its structural information and/or knowledge of previously identified binders. Fewer attempts have been made to develop approaches for de novo design of virtual libraries, as synthesizability of generated molecules remains a challenge. In this work, we developed a new Monte Carlo Search (MCS) algorithm, DrugSynthMC (Drug Synthesis using Monte Carlo), in conjunction with DL and statistical-based priors to generate thousands of interpretable chemical structures and novel drug-like molecules per second. DrugSynthMC produces drug-like compounds using an atom-based search model that builds molecules as SMILES, character by character. Designed molecules follow Lipinski's "rule of 5″, show a high proportion of highly water-soluble nontoxic predicted-to-be synthesizable compounds, and efficiently expand the chemical space within the libraries, without reliance on training data sets, synthesizability metrics, or enforcing during SMILES generation. Our approach can function with or without an underlying neural network and is thus easily explainable and versatile. This ease in drug-like molecule generation allows for future integration of score functions aimed at different target- or job-oriented goals. Thus, DrugSynthMC is expected to enable the functional assessment of large compound libraries covering an extensive novel chemical space, overcoming the limitations of existing drug collections. The software is available at https://github.com/RoucairolMilo/DrugSynthMC.

Developing a Tanshinone IIA Memetic by Targeting MIOS to Regulate mTORC1 and Autophagy in Glioblastoma.

Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy. Recently, T2A has been demonstrated to function through sestrin 2 (SESN) to inhibit mTORC1 activity, but its possible impact on autophagy through this pathway has not been investigated. Here, the model system Dictyostelium discoideum and GBM cell lines were employed to investigate the cellular role of T2A in regulating SESN to inhibit mTORC1 and activate autophagy through a GATOR2 component MIOS. In D. discoideum, T2A treatment induced autophagy and inhibited mTORC1 activity, with both effects lost upon the ablation of SESN (sesn-) or MIOS (mios-). We further investigated the targeting of MIOS to reproduce this effect of T2A, where computational analysis identified 25 novel compounds predicted to strongly bind the human MIOS protein, with one compound (MIOS inhibitor 3; Mi3) reducing cell proliferation in two GBM cells. Furthermore, Mi3 specificity was demonstrated through the loss of potency in the D. discoideum mios- cells regarding cell proliferation and the induction of autophagy. In GBM cells, Mi3 treatment also reduced mTORC1 activity and induced autophagy. Thus, a potential T2A mimetic showing the inhibition of mTORC1 and induction of autophagy in GBM cells was identified.

Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation.

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

Measuring chemical exchange saturation transfer exchange rates in the human brain using a particle swarm optimisation algorithm.

The z-spectrum contains many pools with different exchange rates and T2 values, which can make it difficult to interpret in vivo data and complicates the design of experiments aimed at providing sensitivity to one pool. This work aims to characterise the main pools observable with MRI at 7T in the human brain. To achieve this, we acquired z-spectra at multiple saturation powers in the human brain at 7T. We used simulations to optimise the use of particle swarm optimisation (PSO) to fit these data, validating this approach using further simulations and creatine phantoms. We then used the PSO to fit data from grey and white matter for the pool size, exchange rate, and T2 of five proton pools (magnetisation transfer, amides, amines, nuclear Overhauser enhancement NOE-3.5ppm and NOE-1.7ppm in addition to water). We then devised an approach for using PSO to fit z-spectra while limiting the computational burden, and we investigated the sensitivity of the fit to T2 and k for three overlapping pools. We used this to measure the exchange rate of creatine and to show that it varied with temperature, as expected. In the brain we measured a significantly larger pool size in white matter than in grey matter for the magnetisation transfer pool and the NOE-3.5ppm pool. For all other parameters we found no significant difference between grey and white matter. We showed that PSO can be used to fit z-spectra acquired at a range of B1 to provide information about peak position, amplitude, exchange rate, and T2 in vivo in the human brain. These data could provide more sensitivity to change in some clinical conditions and will also provide key information for further experimental design.

Asymmetric Dimethylation of Ribosomal S6 Kinase 2 Regulates Its Cellular Localisation and Pro-Survival Function.

Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated.

Differential Expression of RSK4 Transcript Isoforms in Cancer and Its Clinical Relevance.

While we previously revealed RSK4 as a therapeutic target in lung and bladder cancers, the wider role of this kinase in other cancers remains controversial. Indeed, other reports instead proposed RSK4 as a tumour suppressor in colorectal and gastric cancers and are contradictory in breast malignancies. One explanation for these discrepancies may be the expression of different RSK4 isoforms across cancers. Four RNAs are produced from the RSK4 gene, with two being protein-coding. Here, we analysed the expression of the latter across 30 normal and 33 cancer tissue types from the combined GTEx/TCGA dataset and correlated it with clinical features. This revealed the expression of RSK4 isoforms 1 and 2 to be independent prognostic factors for patient survival, pathological stage, cancer metastasis, recurrence, and immune infiltration in brain, stomach, cervical, and kidney cancers. However, we found that upregulation of either isoform can equally be associated with good or bad prognosis depending on the cancer type, and changes in the expression ratio of isoforms fail to predict clinical outcome. Hence, differential isoform expression alone cannot explain the contradictory roles of RSK4 in cancers, and further research is needed to highlight the underlying mechanisms for the context-dependent function of this kinase.

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family.

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.

Recurrent patellofemoral instability rates after MPFL reconstruction techniques are in the range of instability rates after other soft tissue realignment techniques.

PURPOSE: Recurrent patellofemoral instability is a common knee injury in skeletally immature patients. Many surgical techniques have been described in the literature, all with different success rates. Purpose of this study was to perform a systematic review and meta-analysis of the available literature to assess recurrent patellofemoral instability rates after surgical treatment using MPFL reconstruction techniques and other soft tissue realignment techniques in skeletally immature patients. METHODS: PubMed, Embase, Web of Science, and The Cochrane Library were searched to identify all original articles concerning the surgical treatment for patellofemoral instability in skeletally immature patients and that reported post-operative recurrent patellofemoral instability rates. Subsequently a risk of bias assessment was conducted and a meta-analysis was performed on reported post-operative recurrent patellofemoral instability rates after MPFL reconstruction techniques and other soft tissue realignment techniques. RESULTS: Of the 21 eligible studies (448 knees in 389 patients), 10 studies reported on MPFL reconstruction techniques using different grafts and fixation techniques and 11 reported on other soft tissue realignment procedures. In total, 62 of the 448 (13.8%) treated knees showed recurrent patellofemoral instability during follow-up. The overall pooled recurrent patellofemoral instability rate was estimated to be 0.08 (95% CI 0.02-0.16). For MPFL reconstruction techniques, the pooled recurrent patellofemoral instability rate was estimated to be 0.02 (95% CI 0.00-0.09). For the other soft tissue realignment techniques, the pooled rate was estimated to be 0.15 (95% CI 0.04-0.31). No statistically significant difference in recurrent patellofemoral instability rates between MPFL reconstruction techniques and other soft tissue realignment techniques were found (n.s.). There was a large variation in treatment effects over different settings, including what effect is to be expected in future patients. CONCLUSION: This systematic review and meta-analysis found that recurrent patellofemoral instability rates after MPFL reconstruction techniques are in the range of instability rates after other soft tissue realignment techniques. The clinical relevance of this study is that it provides clinicians with the best currently available evidence on recurrent patellofemoral instability rates after surgical treatment for patellofemoral instability in skeletally immature patients. LEVEL OF EVIDENCE: IV.

Imaging the atomic structure and local chemistry of platelets in natural type Ia diamond.

In the past decades, many efforts have been devoted to characterizing {001} platelet defects in type Ia diamond. It is known that N is concentrated at the defect core. However, an accurate description of the atomic structure of the defect and the role that N plays in it is still unknown. Here, by using aberration-corrected transmission electron microscopy and electron energy-loss spectroscopy we have determined the atomic arrangement within platelet defects in a natural type Ia diamond and matched it to a prevalent theoretical model. The platelet has an anisotropic atomic structure with a zigzag ordering of defect pairs along the defect line. The electron energy-loss near-edge fine structure of both carbon K- and nitrogen K-edges obtained from the platelet core is consistent with a trigonal bonding arrangement at interstitial sites. The experimental observations support an interstitial aggregate mode of formation for platelet defects in natural diamond.

Relationships between cortical myeloarchitecture and electrophysiological networks.

The human brain relies upon the dynamic formation and dissolution of a hierarchy of functional networks to support ongoing cognition. However, how functional connectivities underlying such networks are supported by cortical microstructure remains poorly understood. Recent animal work has demonstrated that electrical activity promotes myelination. Inspired by this, we test a hypothesis that gray-matter myelin is related to electrophysiological connectivity. Using ultra-high field MRI and the principle of structural covariance, we derive a structural network showing how myelin density differs across cortical regions and how separate regions can exhibit similar myeloarchitecture. Building upon recent evidence that neural oscillations mediate connectivity, we use magnetoencephalography to elucidate networks that represent the major electrophysiological pathways of communication in the brain. Finally, we show that a significant relationship exists between our functional and structural networks; this relationship differs as a function of neural oscillatory frequency and becomes stronger when integrating oscillations over frequency bands. Our study sheds light on the way in which cortical microstructure supports functional networks. Further, it paves the way for future investigations of the gray-matter structure/function relationship and its breakdown in pathology.

Student Self-Efficacy, Classroom Engagement, and Academic Achievement: Comparing Three Theoretical Frameworks.

Student self-efficacy, behavioral engagement, and emotional engagement are key factors for academic achievement. Research has yet to identify the developmental cascades linking these four constructs. Three theoretical frameworks, i.e., Self-Efficacy Theory, the Self-System Model of Motivational Development, and Expectancy-Value Theory, suggest different nexus. Following 671 students (51.8% girls) from their 4th to 6th grade, this study aims to assess competing hypotheses from these three frameworks in math. Three cross-lag models were tested to test each theoretical framework. A fourth and final model was tested to include the significant paths from the previous models. Mediation paths were also tested. Results mainly support assumptions from Self-Efficacy Theory, that is student self-efficacy and academic achievement are mutually associated from 4th to 6th grades. Some of the propositions of Expectancy-Value Theory were also supported. Self-efficacy was associated with later emotional engagement and academic achievement. However, emotional engagement in 5th grade was negatively associated with achievement in 6th grade and was not associated with behavioral engagement. Assumptions from the Self-System Model were not supported by the data. Testing the fourth model revealed an unexpected developmental cascade: 5th-grade self-efficacy mediated the association between 4th-grade achievement and 6th-grade emotional engagement. This last finding may have great implications for young adolescents as emotional engagement is an indicator of student well-being and intrinsic value of learning. Implications for theory validation and intervention targets for adolescents are discussed.

The z-spectrum from human blood at 7T.

Chemical Exchange Saturation Transfer (CEST) has been used to assess healthy and pathological tissue in both animals and humans. However, the CEST signal from blood has not been fully assessed. This paper presents the CEST and nuclear Overhauser enhancement (NOE) signals detected in human blood measured via z-spectrum analysis. We assessed the effects of blood oxygenation levels, haematocrit, cell structure and pH upon the z-spectrum in ex vivo human blood for different saturation powers at 7T. The data were analysed using Lorentzian difference (LD) model fitting and AREX (to compensate for changes in T1), which have been successfully used to study CEST effects in vivo. Full Bloch-McConnell fitting was also performed to provide an initial estimate of exchange rates and transverse relaxation rates of the various pools. CEST and NOE signals were observed at 3.5 ppm, -1.7 ppm and -3.5 ppm and were found to originate primarily from the red blood cells (RBCs), although the amide proton transfer (APT) CEST effect, and NOEs showed no dependence upon oxygenation levels. Upon lysing, the APT and NOE signals fell significantly. Different pH levels in blood resulted in changes in both the APT and NOE (at -3.5 ppm), which suggests that this NOE signal is in part an exchange relayed process. These results will be important for assessing in vivo z-spectra.

miR-515-5p controls cancer cell migration through MARK4 regulation.

Here, we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seq analyses of both oestrogen receptor receptor-positive and receptor-negative breast cancer cells overexpressing miR-515-5p reveal down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3' UTR interaction and that MARK4 knock-down mimics the effect of miR-515-5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation. Furthermore, miR-515-5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR-515-5p/MARK4 regulation in cell migration and metastasis across two common cancers.

Quantitative analysis of the z-spectrum using a numerically simulated look-up table: Application to the healthy human brain at 7T.

PURPOSE: To develop a method that fits a multipool model to z-spectra acquired from non-steady state sequences, taking into account the effects of variations in T1 or B1 amplitude and the results estimating the parameters for a four-pool model to describe the z-spectrum from the healthy brain. METHODS: We compared measured spectra with a look-up table (LUT) of possible spectra and investigated the potential advantages of simultaneously considering spectra acquired at different saturation powers (coupled spectra) to provide sensitivity to a range of different physicochemical phenomena. RESULTS: The LUT method provided reproducible results in healthy controls. The average values of the macromolecular pool sizes measured in white matter (WM) and gray matter (GM) of 10 healthy volunteers were 8.9% ± 0.3% (intersubject standard deviation) and 4.4% ± 0.4%, respectively, whereas the average nuclear Overhauser effect pool sizes in WM and GM were 5% ± 0.1% and 3% ± 0.1%, respectively, and average amide proton transfer pool sizes in WM and GM were 0.21% ± 0.03% and 0.20% ± 0.02%, respectively. CONCLUSIONS: The proposed method demonstrated increased robustness when compared with existing methods (such as Lorentzian fitting and asymmetry analysis) while yielding fully quantitative results. The method can be adjusted to measure other parameters relevant to the z-spectrum. Magn Reson Med 78:645-655, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Stacking disorder in silicon carbide supported cobalt crystallites: an X-ray diffraction, electron diffraction and high resolution electron microscopy study.

Supported cobalt Fischer-Tropsch catalysts are characteristically nanoparticulate and the reduced SiC supported catalyst was found to contain both HCP and FCC polymorphs. This is reflected in the powder XRD patterns and generally there is a poor fit between the experimental and calculated diffractograms. This was ascribed to small crystallite sizes and the occurrence of disorder, manifested as peak broadening and peak shifts. Selected area electron diffraction data of suitably oriented cobalt catalyst grains on silicon carbide supports show non-periodic disorder in the zone axis orientations that contain the common (001) (HCP) and (111) (FCC) reciprocal lattice planes. Both FCC and HCP polymorphs are present in the same grains and these show disorder mainly in the HCP component. The disorder is further examined using high angle annular dark field (HAADF) scanning transmission electron microscopy at atomic resolution and the stacking sequences elucidated. Random sequences of mainly FCC are interrupted by HCP sequences and twin surfaces with reverse stacking sequences are also present. This study highlights the presence of significant disorder in cobalt catalyst grains confirmed by HAADF microscopy.

hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling.

The increased cap-independent translation of anti-apoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylation-dependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its re-import into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCL-XL expression. In short, phosphorylation on novel N-term sites of hnRNPA1 promotes translation of anti-apoptotic proteins and is indispensable for the pro-survival effects of FGF-2.

Histogram analysis of quantitative T1 and MT maps from ultrahigh field MRI in clinically isolated syndrome and relapsing-remitting multiple sclerosis.

This study used quantitative MRI to study normal appearing white matter (NAWM) in patients with clinically isolated syndromes suggestive of multiple sclerosis and relapsing-remitting multiple sclerosis (RRMS). This was done at ultrahigh field (7 T) for greater spatial resolution and sensitivity. 17 CIS patients, 11 RRMS patients, and 20 age-matched healthy controls were recruited. They were scanned using a 3D inversion recovery turbo field echo sequence to measure the longitudinal relaxation time (T1). A 3D magnetization transfer prepared turbo field echo (MT-TFE) sequence was also acquired, first without a presaturation pulse and then with the MT presaturation pulse applied at -1.05 kHz and +1.05 kHz off resonance from water to produce two magnetization transfer ratio maps (MTR(-) and MTR(+)). Histogram analysis was performed on the signal from the voxels in the NAWM mask. The upper quartile cut-off of the T1 histogram was significantly higher in RRMS patients than in controls (p < 0.05), but there was no difference in CIS. In contrast, MTR was significantly different between CIS or RRMS patients and controls (p < 0.05) for most histogram measures considered. The difference between MTR(+) and MTR(-) signals showed that NOE contributions dominated the changes found. There was a weak negative correlation (r = -0.46, p < 0.05) between the mode of T1 distributions and healthy controls' age; this was not significant for MTR(+) (r = -0.34, p > 0.05) or MTR(-) (r = 0.13, p > 0.05). There was no significant correlation between the median of T1, MTR(-), or MTR(+) and the age of healthy controls. Furthermore, no significant correlation was observed between EDSS or disease duration and T1, MTR(-), or MTR(+) for either CIS or RRMS patients. In conclusion, MTR was found to be more sensitive to early changes in MS disease than T1.

Barriers to overlapping complex and general spine surgery at a tertiary academic hospital.

OBJECTIVE: Policy concern and debate surround the concept of overlapping spine surgery. Overlapping surgery specifically refers to nonessential portions of the case or noncutting time overlap. This differs from concurrent surgery, in which critical portions of the procedure overlap. Here the authors explore the barriers for safe and efficient overlapping surgery in academic spinal deformity practice. METHODS: Over a 24-month period, cases of spinal deformity, degenerative cases, anterior cervical discectomy and fusions (ACDFs), and laminectomy were reviewed for duration in operating room (OR) prior to surgery, duration of cutting time, duration in OR after surgery, turnover duration, and time delay from initial start time. Standard degenerative cases were referenced as 1-2 ACDFs as well as 1- to 2-level laminectomy surgery. The blocks of time between two consecutive cutting periods were investigated to determine the feasibility of overlapping an additional surgery. Specifically, the authors compared the blocks of time that include the postsurgery period, the turnover period, and the presurgery period to cutting periods. RESULTS: One hundred twenty-six complex spinal deformity procedures and 85 degenerative cases (including 49 ACDFs and 36 laminectomies) from one center and one neurosurgeon were reviewed. These procedures were performed between September 2019 and December 2021 with a 3-month gap in military deployment. On average, the procedure's duration for cases of deformity was 236.5 minutes, for cases of ACDFs it was 84 minutes, and for cases of laminectomies it was 105.5 minutes. The block of noncutting time while the patient was in the OR showed no difference from the surgical cut time. The turnover time between cases was 52.35 minutes. Of 100 cases scheduled as the first case of the day, 94 had a delay to the OR averaging 18.2 minutes. CONCLUSIONS: The data in this study indicate that estimates for pre- and postsurgical times alone are not sufficient to allow for overlapping surgery. The average cut-time duration of ACDF was 84 minutes; the average presurgical time for deformity was 68 minutes. This highlights the critical analysis for further examination of optimal scheduling, on-time first start, turnover periods, and the orchestration of all members of the providing team to optimize the cutting time for safe and consistent implementation of overlapping spine surgery.

Bed-rest and exercise remobilization: Concurrent adaptations in muscle glucose and protein metabolism.

BACKGROUND: Bed-rest (BR) of only a few days duration reduces muscle protein synthesis and induces skeletal muscle atrophy and insulin resistance, but the scale and juxtaposition of these events have not been investigated concurrently in the same individuals. Moreover, the impact of short-term exercise-supplemented remobilization (ESR) on muscle volume, protein turnover and leg glucose uptake (LGU) in humans is unknown. METHODS: Ten healthy males (24 ± 1 years, body mass index 22.7 ± 0.6 kg/m2) underwent 3 days of BR, followed immediately by 3 days of ESR consisting of 5 × 30 maximal voluntary single-leg isokinetic knee extensions at 90°/s each day. An isoenergetic diet was maintained throughout the study (30% fat, 15% protein and 55% carbohydrate). Resting LGU was calculated from arterialized-venous versus venous difference across the leg and leg blood flow during the steady-state of a 3-h hyperinsulinaemic-euglycaemic clamp (60 mU/m2/min) measured before BR, after BR and after remobilization. Glycogen content was measured in vastus lateralis muscle biopsy samples obtained before and after each clamp. Leg muscle volume (LMV) was measured using magnetic resonance imaging before BR, after BR and after remobilization. Cumulative myofibrillar protein fractional synthetic rate (FSR) and whole-body muscle protein breakdown (MPB) were measured over the course of BR and remobilization using deuterium oxide and 3-methylhistidine stable isotope tracers that were administered orally. RESULTS: Compared with before BR, there was a 45% decline in insulin-stimulated LGU (P < 0.05) after BR, which was paralleled by a reduction in insulin-stimulated leg blood flow (P < 0.01) and removal of insulin-stimulated muscle glycogen storage. These events were accompanied by a 43% reduction in myofibrillar protein FSR (P < 0.05) and a 2.5% decrease in LMV (P < 0.01) during BR, along with a 30% decline in whole-body MPB after 2 days of BR (P < 0.05). Myofibrillar protein FSR and LMV were restored by 3 days of ESR (P < 0.01 and P < 0.01, respectively) but not by ambulation alone. However, insulin-stimulated LGU and muscle glycogen storage were not restored by ESR. CONCLUSIONS: Three days of BR caused concurrent reductions in LMV, myofibrillar protein FSR, myofibrillar protein breakdown and insulin-stimulated LGU, leg blood flow and muscle glycogen storage in healthy, young volunteers. Resistance ESR restored LMV and myofibrillar protein FSR, but LGU and muscle glycogen storage remained depressed, highlighting divergences in muscle fuel and protein metabolism. Furthermore, ambulation alone did not restore LMV and myofibrillar protein FSR in the non-exercised contralateral limb, emphasizing the importance of exercise rehabilitation following even short-term BR.