The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin

AbstractCalsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 μmol Ca2+/mg protein. Apparent Kas for Ca2+ were around 50 μM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under ‘physiological’ conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains-all staining, immunobiolting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS

Reversal of TMS-induced motor twitch by training is associated with a reduction in excitability of the antagonist muscle

Background: A single session of isolated repetitive movements of the thumb can alter the response to transcranial magnetic stimulation (TMS), such that the related muscle twitch measured post-training occurs in the trained direction. This response is attributed to transient excitability changes in primary motor cortex (M1) that form the early part of learning. We investigated; (1) whether this phenomenon might occur for movements at the wrist, and (2) how specific TMS activation patterns of opposing muscles underlie the practice-induced change in direction. Methods: We used single-pulse suprathreshold TMS over the M1 forearm area, to evoke wrist movements in 20 healthy subjects. We measured the preferential direction of the TMS-induced twitch in both the sagittal and coronal plane using an optical goniometer fixed to the dorsum of the wrist, and recorded electromyographic (EMG) activity from the flexor carpi radialis (FCR) and extensor carpi radialis (ECR) muscles. Subjects performed gentle voluntary movements, in the direction opposite to the initial twitch for 5 minutes at 0.2 Hz. We collected motor evoked potentials (MEPs) elicited by TMS at baseline and for 10 minutes after training. Results: Repetitive motor training was sufficient for TMS to evoke movements in the practiced direction opposite to the original twitch. For most subjects the effect of the newly-acquired direction was retained for at least 10 minutes before reverting to the original. Importantly, the direction change of the movement was associated with a significant decrease in MEP amplitude of the antagonist to the trained muscle, rather than an increase in MEP amplitude of the trained muscle. Conclusions: These results demonstrate for the first time that a TMS-twitch direction change following a simple practice paradigm may result from reduced corticospinal drive to muscles antagonizing the trained direction. Such findings may have implications for training paradigms in neurorehabilitation

Rehabilitation robotics: pilot trial of a spatial extension for MIT-Manus

BACKGROUND: Previous results with the planar robot MIT-MANUS demonstrated positive benefits in trials with over 250 stroke patients. Consistent with motor learning, the positive effects did not generalize to other muscle groups or limb segments. Therefore we are designing a new class of robots to exercise other muscle groups or limb segments. This paper presents basic engineering aspects of a novel robotic module that extends our approach to anti-gravity movements out of the horizontal plane and a pilot study with 10 outpatients. Patients were trained during the initial six-weeks with the planar module (i.e., performance-based training limited to horizontal movements with gravity compensation). This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week (total 36 robot treatment sessions). RESULTS: Pilot study demonstrated that the protocol was safe and well tolerated with no patient presenting any adverse effect. Consistent with our past experience with persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy and we have not observed increases in muscle tone or spasticity during the anti-gravity training protocol. Pilot results showed also a reduction in shoulder-elbow impairment following planar horizontal training. Furthermore, it suggested an additional reduction in shoulder-elbow impairment following the anti-gravity training. CONCLUSION: Our clinical experiments have focused on a fundamental question of whether task specific robotic training influences brain recovery. To date several studies demonstrate that in mature and damaged nervous systems, nurture indeed has an effect on nature. The improved recovery is most pronounced in the trained limb segments. We have now embarked on experiments that test whether we can continue to influence recovery, long after the acute insult, with a novel class of spatial robotic devices. This pilot results support the pursuit of further clinical trials to test efficacy and the pursuit of optimal therapy following brain injury

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma

© The Author(s) 2021.[Background]: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers’ ability to metastasise. First anti-metastatic treatments have recently been approved. [Methods]: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. [Results]: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. [Conclusions]: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.GOF’s lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and King’s Health Partners [King’s Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a King’s Health Partners studentship to V.S.M. and G.O.F. V.S.M.’s lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to King’s Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH

Robotic arm rehabilitation in chronic stroke patients with aphasia may promote speech and language recovery (but effect is not enhanced by supplementary tDCS)

Objective: This study aimed to determine the extent to which robotic arm rehabilitation for chronic stroke may promote recovery of speech and language function in individuals with aphasia. Methods: We prospectively enrolled 17 individuals from a hemiparesis rehabilitation study pairing intensive robot assisted therapy with sham or active tDCS and evaluated their speech (N = 17) and language (N = 9) performance before and after a 12-week (36 session) treatment regimen. Performance changes were evaluated with paired t-tests comparing pre- and post-test measures. There was no speech therapy included in the treatment protocol. Results: Overall, the individuals significantly improved on measures of motor speech production from pre-test to post-test. Of the subset who performed language testing (N = 9), overall aphasia severity on a standardized aphasia battery improved from pre-test baseline to post-test. Active tDCS was not associated with greater gains than sham tDCS. Conclusions: This work indicates the importance of considering approaches to stroke rehabilitation across different domains of impairment, and warrants additional exploration of the possibility that robotic arm motor treatment may enhance rehabilitation for speech and language outcomes. Further investigation into the role of tDCS in the relationship of limb and speech/language rehabilitation is required, as active tDCS did not increase improvements over sham tDCS

Associations between circulating interferon and kynurenine/tryptophan pathway metabolites: support for a novel potential mechanism for cognitive dysfunction in SLE

OBJECTIVE: Quinolinic acid (QA), a kynurenine (KYN)/tryptophan (TRP) pathway metabolite, is an N-methyl-D-aspartate receptor agonist that can produce excitotoxic neuron damage. Type I and II interferons (IFNs) stimulate the KYN/TRP pathway, producing elevated QA/kynurenic acid (KA), a potential neurotoxic imbalance that may contribute to SLE-mediated cognitive dysfunction. We determined whether peripheral blood interferon-stimulated gene (ISG) expression associates with elevated serum KYN:TRP and QA:KA ratios in SLE. METHODS: ISG expression (whole-blood RNA sequencing) and serum metabolite ratios (high-performance liquid chromatography) were measured in 72 subjects with SLE and 73 healthy controls (HCs). ISG were identified from published gene sets and individual IFN scores were derived to analyse associations with metabolite ratios, clinical parameters and neuropsychological assessments. SLE analyses were grouped by level of ISG expression ('IFN high', 'IFN low' and 'IFN similar to HC') and level of monocyte-associated gene expression (using CIBERSORTx). RESULTS: Serum KYN:TRP and QA:KA ratios were higher in SLE than in HC (p<0.01). 933 genes were differentially expressed ≥2-fold in SLE versus HC (p<0.05). 70 of the top 100 most highly variant genes were ISG. Approximately half of overexpressed genes that correlated with KYN:TRP and QA:KA ratios (p<0.05) were ISG. In 36 IFN-high subjects with SLE, IFN scores correlated with KYN:TRP ratios (p<0.01), but not with QA:KA ratios. Of these 36 subjects, 23 had high monocyte-associated gene expression, and in this subgroup, the IFN scores correlated with both KY:NTRP and QA:KA ratios (p<0.05). CONCLUSIONS: High ISG expression correlated with elevated KYN:TRP ratios in subjects with SLE, suggesting IFN-mediated KYN/TRP pathway activation, and with QA:KA ratios in a subset with high monocyte-associated gene expression, suggesting that KYN/TRP pathway activation may be particularly important in monocytes. These results need validation, which may aid in determining which patient subset may benefit from therapeutics directed at the IFN or KYN/TRP pathways to ameliorate a potentially neurotoxic QA/KA imbalance

XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development

The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1fl/fl CD19+/cre conditional knockout (XBP1CD19) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1CD19 mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1CD19 mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment

Accurate prediction of clinical stroke scales and improved biomarkers of motor impairment from robotic measurements

Objective: One of the greatest challenges in clinical trial design is dealing with the subjectivity and variability introduced by human raters when measuring clinical end-points. We hypothesized that robotic measures that capture the kinematics of human movements collected longitudinally in patients after stroke would bear a significant relationship to the ordinal clinical scales and potentially lead to the development of more sensitive motor biomarkers that could improve the efficiency and cost of clinical trials. Materials and methods: We used clinical scales and a robotic assay to measure arm movement in 208 patients 7, 14, 21, 30 and 90 days after acute ischemic stroke at two separate clinical sites. The robots are low impedance and low friction interactive devices that precisely measure speed, position and force, so that even a hemiparetic patient can generate a complete measurement profile. These profiles were used to develop predictive models of the clinical assessments employing a combination of artificial ant colonies and neural network ensembles. Results: The resulting models replicated commonly used clinical scales to a cross-validated R2 of 0.73, 0.75, 0.63 and 0.60 for the Fugl-Meyer, Motor Power, NIH stroke and modified Rankin scales, respectively. Moreover, when suitably scaled and combined, the robotic measures demonstrated a significant increase in effect size from day 7 to 90 over historical data (1.47 versus 0.67). Discussion and conclusion: These results suggest that it is possible to derive surrogate biomarkers that can significantly reduce the sample size required to power future stroke clinical trials

Constitutive Vagus Nerve Activation Modulates Immune Suppression in Sepsis Survivors

Patients surviving a septic episode exhibit persistent immune impairment and increased mortality due to enhanced vulnerability to infections. In the present study, using the cecal ligation and puncture (CLP) model of polymicrobial sepsis, we addressed the hypothesis that altered vagus nerve activity contributes to immune impairment in sepsis survivors. CLP-surviving mice exhibited less TNFα in serum following administration of LPS, a surrogate for an infectious challenge, than control-operated (control) mice. To evaluate the role of the vagus nerve in the diminished response to LPS, mice were subjected to bilateral subdiaphragmatic vagotomy at 2 weeks post-CLP. CLP-surviving vagotomized mice exhibited increased serum and tissue TNFα levels in response to LPS-challenge compared to CLP-surviving, non-vagotomized mice. Moreover, vagus nerve stimulation in control mice diminished the LPS-induced TNFα responses while having no effect in CLP mice, suggesting constitutive activation of vagus nerve signaling in CLP-survivors. The percentage of splenic CD4+ ChAT-EGFP+ T cells that relay vagus signals to macrophages was increased in CLP-survivors compared to control mice, and vagotomy in CLP-survivors resulted in a reduced percentage of ChAT-EGFP+ cells. Moreover, CD4 knockout CLP-surviving mice exhibited an enhanced LPS-induced TNFα response compared to wild-type mice, supporting a functional role for CD4+ ChAT+ T cells in mediating inhibition of LPS-induced TNFα responses in CLP-survivors. Blockade of the cholinergic anti-inflammatory pathway with methyllcaconitine, an α7 nicotinic acetylcholine receptor antagonist, restored LPS-induced TNFα responses in CLP-survivors. Our study demonstrates that the vagus nerve is constitutively active in CLP-survivors and contributes to the immune impairment