Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation.

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Nerve growth factor plays a role in the neurotherapeutic effect of a CD45+ pan-hematopoietic subpopulation derived from human umbilical cord blood in a traumatic brain injury model.

BACKGROUND AIMS: Human umbilical cord blood (HUCB) is an important source of stem cells for therapy of hematopoietic disorders and is a potential therapy for various neurological disorders, including traumatic brain injury (TBI). The expression of nerve growth factor (NGF) and its receptors TrkA, p75NTR and α9ß1 integrin on an HUCB CD45+ pan-hematopoietic subpopulation was investigated in the context of its neurotherapeutic potential after TBI. METHODS: NGF and its receptors were detected on CD45+ cells by reverse transcriptase polymerase chain reaction, flow cytometry analysis and confocal microscopy. CD45+ cells were stimulated by TBI brain extracts, and NGF levels were measured by enzyme-linked immunosorbent assay. TBI mice were divided into six groups for xenogeneic intravenous transplantation, 1 day post-trauma, with 1 × 106 CD45+ cells untreated or treated with the anti-NGF neutralizing antibody K252a, a TrkA antagonist; VLO5, an α9ß1 disintegrin; or negative (vehicle) and positive (NGF) controls. RESULTS: The HUCB CD45+ subpopulation constitutively expresses NGF and its receptors, mainly TrkA and p75NTR and minor levels of α9ß1. In vitro experiments provided evidence that trauma-related mediators from brain extracts of TBI mice induced release of NGF from HUCB CD45+ cell cultures. HUCB CD45+ cells induced a neurotherapeutic effect in TBI mice, abrogated by cell treatment with either anti-NGF antibody or K252a, but not VLO5. CONCLUSIONS: These findings strengthen the role of NGF and its TrkA receptor in the HUCB CD45+ subpopulation's neurotherapeutic effect. The presence of neurotrophin receptors in the HUCB CD45+ pan-hematopoietic subpopulation may explain the neuroprotective effect of cord blood in therapy of a variety of neurological disorders.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease.

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila.

Obscurin (also known as Unc-89 in Drosophila) is a large modular protein in the M-line of Drosophila muscles. Drosophila obscurin is similar to the nematode protein UNC-89. Four isoforms are found in the muscles of adult flies: two in the indirect flight muscle (IFM) and two in other muscles. A fifth isoform is found in the larva. The larger IFM isoform has all the domains that were predicted from the gene sequence. Obscurin is in the M-line throughout development of the embryo, larva and pupa. Using P-element mutant flies and RNAi knockdown flies, we have investigated the effect of decreased obscurin expression on the structure of the sarcomere. Embryos, larvae and pupae developed normally. In the pupa, however, the IFM was affected. Although the Z-disc was normal, the H-zone was misaligned. Adults were unable to fly and the structure of the IFM was irregular: M-lines were missing and H-zones misplaced or absent. Isolated thick filaments were asymmetrical, with bare zones that were shifted away from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depends on the symmetry of adjacent thick filaments; shifted bare zones resulted in abnormally long or short thin filaments. We conclude that obscurin in the IFM is necessary for the development of a symmetrical sarcomere in Drosophila IFM.

Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro.

Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.

A conditional gating mechanism assures the integrity of the molecular force-sensor titin kinase.

As more and more recent investigations point out, force plays an important role in cellular regulation mechanisms. Biological responses to mechanical stress are often based on force-induced conformational changes of single molecules. The force sensor, titin kinase, is involved in a signaling complex that regulates protein turnover and transcriptional adaptation in striated muscle. The structural architecture of such a force sensor determines its response to force and must assure both activity and mechanical integrity, which are prerequisites for its function. Here, we use single-molecule force-clamp spectroscopy to show that titin kinase is organized in such a way that the regulatory domains have to unfold before secondary structure elements that determine the overall fold and catalytic function. The stepwise unfolding over many barriers with a topologically determined sequence assures that the protein can react to force by conformational changes while maintaining its structural integrity.

Regional sensitivity to neuroinflammation: in vivo and in vitro studies.

BACKGROUND: Neuroinflammation is involved in several acute-onset neuropathologies such as meningitis, encephalitis, stroke, and traumatic brain injury as well as in neurodegenerative diseases. All of these patholologies are associated with cognitive deficits. Using a model of pure neuroinflammation (intracisternal injection of endotoxin in mice), we tested the hypothesis that brain regions involved in cognition are the most vulnerable to inflammatory insults, and this vulnerability is an inherent property of neocortical neurons. METHODS: Mice (n = 10/group) injected with endotoxin (LPS) or saline in the cisterna magna underwent neurobehavioral and cognitive testing followed by quantitative autoradiographic assessment of regional neuroinflammation with [3H]PK11195, an established marker of microgliosis. In parallel, cocultures of cortical and striatal neurons taken from embryonic day 19 rat embryos or postnatal day 1 mice expressing green fluorescent protein were exposed for 24 h to the proinflammatory cytokine TNFalpha, glutamate, or a combination of the two agents. RESULTS: LPS-treated mice exhibited significant deficits in memory and significant increases in specific PK11195 binding in cortical and hippocampal regions, but not in striatum. Cultured neurons of cortical origin showed significantly lower survival rate relative to striatal neurons in response to TNFalpha, glutamate, or a combination of the two agents. Furthermore, TNFalpha exerted neuroprotective rather than neurotoxic effects in the striatal but not in the cortical neurons. CONCLUSIONS: These results suggest that the cortex is inherently more sensitive than the striatum to the deleterious effects of neuroinflammation, and may offer an explanation for the preponderance of cognitive deficits in neuropathologies with a neuroinflammatory component.

Mechanoenzymatics of titin kinase.

Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.

Molecular noise filtering in the ß-adrenergic signaling network by phospholamban pentamers.

Phospholamban (PLN) is an important regulator of cardiac calcium handling due to its ability to inhibit the calcium ATPase SERCA. ß-Adrenergic stimulation reverses SERCA inhibition via PLN phosphorylation and facilitates fast calcium reuptake. PLN also forms pentamers whose physiological significance has remained elusive. Using mathematical modeling combined with biochemical and cell biological experiments, we show that pentamers regulate both the dynamics and steady-state levels of monomer phosphorylation. Substrate competition by pentamers and a feed-forward loop involving inhibitor-1 can delay monomer phosphorylation by protein kinase A (PKA), whereas cooperative pentamer dephosphorylation enables bistable PLN steady-state phosphorylation. Simulations show that phosphorylation delay and bistability act as complementary filters that reduce the effect of random fluctuations in PKA activity, thereby ensuring consistent monomer phosphorylation and SERCA activity despite noisy upstream signals. Preliminary analyses suggest that the PLN mutation R14del could impair noise filtering, offering a new perspective on how this mutation causes cardiac arrhythmias.

High Throughput Screen Identifies Small Molecule Effectors That Modulate Thin Filament Activation in Cardiac Muscle.

Current therapeutic interventions for both heart disease and heart failure are largely insufficient and associated with undesired side effects. Biomedical research has emphasized the role of sarcomeric protein function for the normal performance and energy efficiency of the heart, suggesting that directly targeting the contractile myofilaments themselves using small molecule effectors has therapeutic potential and will likely result in greater drug efficacy and selectivity. In this study, we developed a robust and highly reproducible fluorescence polarization-based high throughput screening (HTS) assay that directly targets the calcium-dependent interaction between cardiac troponin C (cTnC) and the switch region of cardiac troponin I (cTnISP), with the aim of identifying small molecule effectors of the cardiac thin filament activation pathway. We screened a commercially available small molecule library and identified several hit compounds with both inhibitory and activating effects. We used a range of biophysical and biochemical methods to characterize hit compounds and identified fingolimod, a sphingosin-1-phosphate receptor modulator, as a new troponin-based small molecule effector. Fingolimod decreased the ATPase activity and calcium sensitivity of demembranated cardiac muscle fibers in a dose-dependent manner, suggesting that the compound acts as a calcium desensitizer. We investigated fingolimod's mechanism of action using a combination of computational studies, biophysical methods, and synthetic chemistry, showing that fingolimod bound to cTnC repels cTnISP via mainly electrostatic repulsion of its positively charged tail. These results suggest that fingolimod is a potential new lead compound/scaffold for the development of troponin-directed heart failure therapeutics.

Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury.

Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan-histone deacetylase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h postinjury. Using a well-characterized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neurobehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P< or =0.05), and that this functional benefit is accompanied by decreased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P< or =0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury-induced decreases in cytoprotective heat-shock protein 70 kDa and phosphorylated Akt. Moreover, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase-3 immunoreactivity within microglia/macrophages at the trauma area were elevated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our findings underscore the relevance of HDAC inhibitors for ameliorating trauma-induced functional deficits and warrant consideration of applying ITF2357 for this indication.

A role for GATA-6 in vertebrate chondrogenesis.

The GATA family of transcription factors are known to play multiple critical roles in vertebrate developmental processes, including erythropoiesis, endoderm formation and cardiogenesis. There have been no previous demonstrations of a functional role for any GATA family member being associated with musculoskeletal development but we now identify a possible role for GATA-6 in chondrogenesis. We detect abundant levels of GATA-6 mRNA in precartilaginous condensations (PCCs) in both the axial and appendicular skeleton of mouse embryos and in committed primary chondrocyte precursors. We also show that the G-protein coupled receptor, Gpr49, is a target of GATA-6 regulation in differentiating embryonal carcinoma cells and that, in vivo, the expression domains of the two genes overlap within PCCs. Finally, we have identified conserved, canonical GATA binding sites within the Gpr49 gene locus, and show by EMSAs that GATA-6 can bind to these sites in vitro. These data therefore suggest that GATA-6 also plays a role in chondrogenesis and that Gpr49 is a potential direct target of GATA regulation in this process.

Dynamic changes in the recovery after traumatic brain injury in mice: effect of injury severity on T2-weighted MRI abnormalities, and motor and cognitive functions.

Memory and neurobehavioral dysfunctions are among the sequelae of traumatic brain injury (TBI). The Neurological Severity Score (NSS) includes 10 tasks and was previously designed to assess the functional status of mice after TBI. The object recognition task (ORT) measures specific episodic memory and is expressed by the percent time spent by an animal at a novel, unfamiliar object (Discrimination Index [DI]). It is an ideal tool for evaluating cognitive function after TBI. The present study sought to validate the use of the NSS and ORT in severe and mild focal TBI in mice, and to confirm that the spontaneous recovery and the radiological abnormalities, shown by T2-weighted magnetic resonance imaging (MRI), are dependent upon injury severity. Mice were subjected to severe and mild closed head injury (NSS at 1 h 7.52 +/- 0.34 and 4.62 +/- 0.14, respectively). NSS was evaluated for 25 days and showed a decrease by 3.86 +/- 0.26 and 2.54 +/- 0.35 units in the severely and mildly injured mice, respectively. ORT revealed DI in severely injured group of 51.7 +/- 6.15%, (vs approximately 75-80% in uninjured animal) on day 3 and 66.2 +/- 6.81% on day 21. In contrast, the mildly injured mice did not show cognitive impairment throughout the same period. The damage seen by MRI at 24 h after injury, strongly correlated with NSS(1h) (R = 0.87, p < 0.001). We conclude that NSS is a reliable tool for evaluation of neurological damage in head-injured mice, NSS(1h) predicts the motor dysfunction, cognitive damage, and brain-damage characteristics as depicted by T2-weighted MRI. The combined assessment of neurobehavioral and cognitive function along with MRI is most useful in evaluating recovery from injury, especially when testing effectiveness of novel treatments or genetic manipulations.

Differential neuroprotective properties of endogenous and exogenous erythropoietin in a mouse model of traumatic brain injury.

Both heat acclimation (HA) and post-injury treatment with recombinant human erythropoietin (Epo, rhEpo, exogenous Epo) are neuroprotective against traumatic brain injury (TBI). Our previous data demonstrated that HA-induced neuroprotection includes improved functional recovery and reduced cerebral edema formation. Additionally, in earlier Western-blot analyses, we found that HA mice display increased expression of the specific erythropoietin receptor (EpoR) and of hypoxia-inducible factor-1 alpha (HIF-1 alpha), the inducible subunit of the transcription factor, which regulates Epo gene expression, but not of Epo itself. In light of this, the aim of the current study was threefold: (1) to assess Epo expression in the trauma area and hippocampus following HA, rhEpo administration, or combined HA-rhEpo treatment, using immunohistochemical methods that offer enhanced anatomical resolution; (2) to examine the effects of endogenous and exogenous Epo on edema formation in normothermic (NT) mice; and (3) to evaluate the effects of exogenous Epo administration on neuroprotective outcome measures in HA animals. HA induced enhanced expression of endogenous Epo in the trauma area and the hippocampus. Treatment with anti-Epo antibody given to NT mice increased edema formation, whereas rhEpo induced no beneficial effect. Cognitive performance testing and immunohistochemical findings reinforced HA and rhEpo as separate protective interventions but showed no advantage to combining the two strategies. We therefore suggest that HA-induced neuroprotection is shaped by pre-existing mediators but cannot be modified by post-injury treatment aimed at increasing the levels of neuroprotective agents.

D-cycloserine improves functional recovery and reinstates long-term potentiation (LTP) in a mouse model of closed head injury.

Traumatic brain injury triggers a massive glutamate efflux, activation of NMDA receptor channels, and cell death. Recently, we reported that NMDA receptors in mice are down-regulated from hours to days following closed head injury (CHI), and treatment with NMDA improved recovery of motor and cognitive functions up to 14 d post-injury. Here we show that a single injection of a low dose of D-cycloserine (DCS), a partial NMDA receptor agonist, in CHI mice 24 h post-injury, resulted in a faster and greater recovery of motor and memory functions as assessed by neurological severity score and object recognition tests, respectively. Moreover, DCS treatment of CHI mice led to a significant improvement of hippocampal long-term potentiation (LTP) in the CA1 region that was completely blunted in CHI control mice. However, DCS did not improve CHI-induced impairment in synaptic glutamate release measured by paired pulse facilitation (PPF) ratio in hippocampal CA1 region. Finally, CHI-induced reduction of brain-derived neurotrophic factor (BDNF) was fully restored following DCS treatment. Since DCS is in clinical use for other indications, the present study offers a novel approach to treat human brain injury.

Akt phosphorylation is required for heat acclimation-induced neuroprotection.

Long-term heat exposure, known as heat acclimation (HA; 30 days at 34 +/- 1 degrees C) is neuroprotective against traumatic brain injury. Acclimated mice were previously found to display improved functional recovery as well as an increase in the levels of the specific erythropoietin receptor. As the activation of this receptor is known to facilitate functional recovery on one hand and the phosphorylation and activation of Akt, an intracellular kinase which regulates anti-apoptotic pathways on the other, in this study we investigated whether HA affects Akt phosphorylation prior to and following injury and whether this step is required for development of HA-induced neuroprotection. Akt phosphorylation was blocked using Triciribine (TCN), a compound shown to block the phosphorylation process without affecting upstream effectors of this kinase, and several post-injury functional end-point measures were subsequently evaluated. Acclimation led to a post-injury increase in the levels of phosphorylated Akt, resulting in higher levels when compared with normothermic controls at 4 h post-injury (63.6 +/- 5.2% and 42.7 +/- 3.7%, respectively, p

Wnt2 is a direct downstream target of GATA6 during early cardiogenesis.

The GATA4, 5 and 6 subfamily of transcription factors are potent transactivators of transcription expressed within the precardiac mesoderm. However, little is known of the immediate downstream targets of GATA-factor regulation during the earliest stages of cardiogenesis. Using the P19-CL6 embryonal carcinoma (EC) cell line as an in vitro model of cardiogenesis, we show that GATA6 is the most abundantly expressed of the GATA factors in presumptive cardiac cells. Consequently, we performed a microarray screen comparing mRNA from control EC cells, early in the cardiac differentiation pathway, with those in which GATA6 had been overexpressed. These studies identified 103 genes whose expression changed significantly and this was verified in a representative array of these genes by real-time RT-PCR. We show that early cardiac expression of one of these genes, Wnt2, mirrors that of GATA6 in vitro and in vivo. In addition, its upregulation by GATA6 in differentiating EC cells is mediated by the direct binding of GATA-factor(s) to the cognate Wnt2 promoter, suggesting Wnt2 is an immediate downstream target of GATA-factor regulation during early cardiogenesis.

low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits.

Low-level laser therapy (LLLT) has been evaluated in this study as a potential therapy for traumatic brain injury (TBI). LLLT has been found to modulate various biological processes. Following TBI in mice, we assessed the hypothesis that LLLT might have a beneficial effect on their neurobehavioral and histological outcome. TBI was induced by a weight-drop device, and motor function was assessed 1 h post-trauma using a neurological severity score (NSS). Mice were then divided into three groups of eight mice each: one control group that received a sham LLLT procedure and was not irradiated; and two groups that received LLLT at two different doses (10 and 20 mW/cm(2) ) transcranially. An 808-nm Ga-As diode laser was employed transcranially 4 h post-trauma to illuminate the entire cortex of the brain. Motor function was assessed up to 4 weeks, and lesion volume was measured. There were no significant changes in NSS at 24 and 48 h between the laser-treated and non-treated mice. Yet, from 5 days and up to 28 days, the NSS of the laser-treated mice were significantly lower (p < 0.05) than the traumatized control mice that were not treated with the laser. The lesion volume of the laser treated mice was significantly lower (1.4%) than the non-treated group (12.1%). Our data suggest that a non-invasive transcranial application of LLLT given 4 h following TBI provides a significant long-term functional neurological benefit. Further confirmatory trials are warranted.

Altered cytokine expression and sustained hypothermia following traumatic brain injury in heat acclimated mice.

Long-term exposure to moderate ambient heat (heat acclimation, HA, 30 days at 34+/-1 degrees C) provides protection toward a variety of stressors including traumatic brain injury. As previous studies suggested an anti-inflammatory effect of HA and given the ability of augmented pre-injury anti-inflammatory cytokine expression to harbor neuroprotection and to attenuate early post-injury expression of pro-inflammatory mediators, we hypothesized that HA-induced neuroprotection may involve enhanced pre-injury expression of anti-inflammatory mediators or a reduction in post-injury TNF alpha (TNFalpha) expression. Since the attenuation of inflammatory-associated entities has also been linked to mild hypothermia, an established neuroprotective paradigm, the effect of HA on post-injury body temperature was also studied. HA mice and normothermic (NT) counterparts were examined using a closed head injury model. Cytokines were measured within the ipsilateral cortex. Pre-injury protein levels of anti-inflammatory interleukins 10 and 4 (IL-10, IL-4) were quantified by enzyme-linked immunosorbent assays (ELISA). mRNA and protein levels of TNFalpha were quantified during the initial 2 h post-injury using semi-quantitative and real-time polymerase chain reaction (sqRT-PCR and qRT-PCR) or ELISA, respectively. Rectal temperatures were measured. HA induced augmented pre-injury IL-10 expression and a post-injury reduction in TNFalpha mRNA levels, as well as altered expression dynamics of TNFalpha protein. TNFalpha protein levels decreased relative to the sham state in HA mice only. HA mice displayed sustained post-injury hypothermia, namely significantly lower body temperature at 4 h post-injury. Given the evidence on the neuroprotective nature of hypothermia and anti-inflammatory cytokines, we suggest that these changes may contribute to HA-induced neuroprotection.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase.

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.