Concussion susceptibility is mediated by spreading depolarization-induced neurovascular dysfunction.

The mechanisms underlying the complications of mild traumatic brain injury, including post-concussion syndrome, post-impact catastrophic death, and delayed neurodegeneration remain poorly understood. This limited pathophysiological understanding has hindered the development of diagnostic and prognostic biomarkers and has prevented the advancement of treatments for the sequelae of mild traumatic brain injury. We aimed to characterize the early electrophysiological and neurovascular alterations following repetitive mild traumatic brain injury and sought to identify new targets for the diagnosis and treatment of individuals at risk of severe post-impact complications. We combined behavioural, electrophysiological, molecular, and neuroimaging techniques in a rodent model of repetitive mild traumatic brain injury. In humans, we used dynamic contrast-enhanced MRI to quantify blood-brain barrier dysfunction after exposure to sport-related concussive mild traumatic brain injury. Rats could clearly be classified based on their susceptibility to neurological complications, including life-threatening outcomes, following repetitive injury. Susceptible animals showed greater neurological complications and had higher levels of blood-brain barrier dysfunction, transforming growth factor ß (TGFß) signalling, and neuroinflammation compared to resilient animals. Cortical spreading depolarizations were the most common electrophysiological events immediately following mild traumatic brain injury and were associated with longer recovery from impact. Triggering cortical spreading depolarizations in mild traumatic brain injured rats (but not in controls) induced blood-brain barrier dysfunction. Treatment with a selective TGFß receptor inhibitor prevented blood-brain barrier opening and reduced injury complications. Consistent with the rodent model, blood-brain barrier dysfunction was found in a subset of human athletes following concussive mild traumatic brain injury. We provide evidence that cortical spreading depolarization, blood-brain barrier dysfunction, and pro-inflammatory TGFß signalling are associated with severe, potentially life-threatening outcomes following repetitive mild traumatic brain injury. Diagnostic-coupled targeting of TGFß signalling may be a novel strategy in treating mild traumatic brain injury.

Attenuated CSF-1R signalling drives cerebrovascular pathology.

Cerebrovascular pathologies occur in up to 80% of cases of Alzheimer's disease; however, the underlying mechanisms that lead to perivascular pathology and accompanying blood-brain barrier (BBB) disruption are still not fully understood. We have identified previously unreported mutations in colony stimulating factor-1 receptor (CSF-1R) in an ultra-rare autosomal dominant condition termed adult-onset leucoencephalopathy with axonal spheroids and pigmented glia (ALSP). Cerebrovascular pathologies such as cerebral amyloid angiopathy (CAA) and perivascular p-Tau were some of the primary neuropathological features of this condition. We have identified two families with different dominant acting alleles with variants located in the kinase region of the CSF-1R gene, which confer a lack of kinase activity and signalling. The protein product of this gene acts as the receptor for 2 cognate ligands, namely colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). Here, we show that depletion in CSF-1R signalling induces BBB disruption and decreases the phagocytic capacity of peripheral macrophages but not microglia. CSF-1R signalling appears to be critical for macrophage and microglial activation, and macrophage localisation to amyloid appears reduced following the induction of Csf-1r heterozygosity in macrophages. Finally, we show that endothelial/microglial crosstalk and concomitant attenuation of CSF-1R signalling causes re-modelling of BBB-associated tight junctions and suggest that regulating BBB integrity and systemic macrophage recruitment to the brain may be therapeutically relevant in ALSP and other Alzheimer's-like dementias.

Finite element simulation of head impacts in mixed martial arts.

Thirteen MMA athletes were fitted with the MiG2.0 Stanford instrumented mouthguard. 451 video confirmed impacts were recoded during sparring sessions and competitive events. The competitive events resulted in five concussions. The impact with the highest angular acceleration from each event was simulated using the GHBMC head model. Average strain in the corpus callosum of concussed fighters was 0.27, which was 87.9% higher than uninjured fighters and was the best strain indicator of concussion. The best overall predictor of concussion found in this study was shear stress in the corpus callosum which differed by 111.4% between concussed and uninjured athletes.

Concussion and the severity of head impacts in mixed martial arts.

Concern about the consequences of head impacts in US football has motivated researchers to investigate and develop instrumentation to measure the severity of these impacts. However, the severity of head impacts in unhelmeted sports is largely unknown as miniaturised sensor technology has only recently made it possible to measure these impacts in vivo. The objective of this study was to measure the linear and angular head accelerations in impacts in mixed martial arts, and correlate these with concussive injuries. Thirteen mixed martial arts fighters were fitted with the Stanford instrumented mouthguard (MiG2.0) participated in this study. The mouthguard recorded linear acceleration and angular velocity in 6 degrees of freedom. Angular acceleration was calculated by differentiation. All events were video recorded, time stamped and reported impacts confirmed. A total of 451 verified head impacts above 10g were recorded during 19 sparring events (n = 298) and 11 competitive events (n = 153). The average resultant linear acceleration was 38.0624.3g while the average resultant angular acceleration was 256761739 rad/s2. The competitive bouts resulted in five concussions being diagnosed by a medical doctor. The average resultant acceleration (of the impact with the highest angular acceleration) in these bouts was 86.7618.7g and 756163438 rad/s2. The average maximum Head Impact Power was 20.6kW in the case of concussion and 7.15kW for the uninjured athletes. In conclusion, the study recorded novel data for sub-concussive and concussive impacts. Events that resulted in a concussion had an average maximum angular acceleration that was 24.7% higher and an average maximum Head Impact Power that was 189% higher than events where there was no injury. The findings are significant in understanding the human tolerance to short-duration, high linear and angular accelerations.

Multi-Directional Dynamic Model for Traumatic Brain Injury Detection.

Given the worldwide adverse impact of traumatic brain injury (TBI) on the human population, its diagnosis and prediction are of utmost importance. Historically, many studies have focused on associating head kinematics to brain injury risk. Recently, there has been a push toward using computationally expensive finite element (FE) models of the brain to create tissue deformation metrics of brain injury. Here, we develop a new brain injury metric, the brain angle metric (BAM), based on the dynamics of a 3 degree-of-freedom lumped parameter brain model. The brain model is built based on the measured natural frequencies of an FE brain model simulated with live human impact data. We show that it can be used to rapidly estimate peak brain strains experienced during head rotational accelerations that cause mild TBI. In our data set, the simplified model correlates with peak principal FE strain (R2 = 0.82). Further, coronal and axial brain model displacement correlated with fiber-oriented peak strain in the corpus callosum (R2 = 0.77). Our proposed injury metric BAM uses the maximum angle predicted by our brain model and is compared against a number of existing rotational and translational kinematic injury metrics on a data set of head kinematics from 27 clinically diagnosed injuries and 887 non-injuries. We found that BAM performed comparably to peak angular acceleration, translational acceleration, and angular velocity in classifying injury and non-injury events. Metrics that separated time traces into their directional components had improved model deviance compare with those that combined components into a single time trace magnitude. Our brain model can be used in future work to rapidly approximate the peak strain resulting from mild to moderate head impacts and to quickly assess brain injury risk.

Dynamic Blood-Brain Barrier Regulation in Mild Traumatic Brain Injury.

Whereas the diagnosis of moderate and severe traumatic brain injury (TBI) is readily visible on current medical imaging paradigms (magnetic resonance imaging [MRI] and computed tomography [CT] scanning), a far greater challenge is associated with the diagnosis and subsequent management of mild TBI (mTBI), especially concussion which, by definition, is characterized by a normal CT. To investigate whether the integrity of the blood-brain barrier (BBB) is altered in a high-risk population for concussions, we studied professional mixed martial arts (MMA) fighters and adolescent rugby players. Additionally, we performed the linear regression between the BBB disruption defined by increased gadolinium contrast extravasation on dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) on MRI and multiple biomechanical parameters indicating the severity of impacts recorded using instrumented mouthguards in professional MMA fighters. MMA fighters were examined pre-fight for a baseline and again within 120 h post-competitive fight, whereas rugby players were examined pre-season and again post-season or post-match in a subset of cases. DCE-MRI, serological analysis of BBB biomarkers, and an analysis of instrumented mouthguard data, was performed. Here, we provide pilot data that demonstrate disruption of the BBB in both professional MMA fighters and rugby players, dependent on the level of exposure. Our data suggest that biomechanical forces in professional MMA and adolescent rugby can lead to BBB disruption. These changes on imaging may serve as a biomarker of exposure of the brain to repetitive subconcussive forces and mTBI.

Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production.

Bacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain, Lactobacillus plantarum NI326, with activity against the beverage-spoilage bacterium Alicyclobacillus acidoterrestris Genome sequencing of NI326 enabled the identification of a gene cluster (designated plc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant of Lb. plantarum NI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. The plc gene cluster was cloned and expressed in Lactococcus lactis NZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced by Lb. plantarum The inhibition displayed against A. acidoterrestris highlights its potential use as a preservative in food and beverages.IMPORTANCE In this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by a Lactobacillus plantarum strain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, including Alicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for a Lactobacillus plantarum strain.

A role for Rab14 in the endocytic trafficking of GLUT4 in 3T3-L1 adipocytes.

Insulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged 'ring-like' vesicular structures (mean diameter 1.3 µm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

The Rab family of proteins: 25 years on.

Intracellular membrane trafficking requires the complex interplay of several classes of trafficking proteins. Rab proteins, the largest subfamily of the Ras superfamily of small G-proteins, are central regulators of all aspects of intracellular trafficking processes including vesicle budding and uncoating, motility, tethering and fusion. In the present paper, we discuss the discovery, evolution and characterization of the Rab GTPase family. We examine their basic functional roles, their important structural features and the regulatory proteins which mediate Rab function. We speculate on outstanding issues in the field, such as the mechanisms of Rab membrane association and the co-ordinated interplay between distinct Rab proteins. Finally, we summarize the data implicating Rab proteins in an ever increasing number of diseases.

Rab11 proteins in health and disease.

Comprising over 60 members, Rab proteins constitute the largest branch of the Ras superfamily of low-molecular-mass G-proteins. This protein family have been primarily implicated in various aspects of intracellular membrane trafficking processes. On the basis of distinct subfamily-specific sequence motifs, many Rabs have been grouped into subfamilies. The Rab11 GTPase subfamily comprises three members: Rab11a, Rab11b and Rab25/Rab11c, which, between them, have been demonstrated to bind more than 30 proteins. In the present paper, we review the function of the Rab11 subfamily. We describe their localization and primary functional roles within the cell and their implication, to date, in disease processes. We also summarize the protein machinery currently known to regulate or mediate their functions and the cargo molecules which they have been shown to transport.

Tumor susceptibility gene 101 (TSG101) is a novel binding-partner for the class II Rab11-FIPs.

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.

Rab30 is required for the morphological integrity of the Golgi apparatus.

BACKGROUND INFORMATION: Rab GTPases are key coordinators of eukaryotic intracellular membrane trafficking. In their active states, Rabs localise to the cytoplasmic face of intracellular compartments where they regulate membrane trafficking processes. Many Rabs have been extensively characterised whereas others, such as Rab30, have to date received relatively little attention. RESULTS: Here, we demonstrate that Rab30 is primarily associated with the secretory pathway, displaying predominant localisation to the Golgi apparatus. We find by time-lapse microscopy and fluorescence recovery after photobleaching studies that Rab30 is rapidly and continuously recruited to the Golgi. We also show that Rab30 function is required for the morphological integrity of the Golgi. Finally, we demonstrate that inactivation of Rab30 does not impair anterograde or retrograde transport through the Golgi. CONCLUSIONS: Taken together, these data illustrate that Rab30 primarily localises to the Golgi apparatus and is required for the structural integrity of this organelle.

The role of endosomal-recycling in long-term potentiation.

Long-term potentiation (LTP) defines persistent increases in neurotransmission strength at synapses that are triggered by specific patterns of neuronal activity. LTP, the most widely accepted molecular model for learning, is best characterised at glutamatergic synapses on dendritic spines. In this context, LTP involves increases in dendritic spine size and the insertion of glutamate receptors into the post-synaptic spine membrane, which together boost post-synaptic responsiveness to neurotransmitters. In dendrites, the material required for LTP is sourced from an organelle termed the endosomal-recycling compartment (ERC), which is localised to the base of dendritic spines. When LTP is induced, material derived from the recycling compartment, which contains α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs), is mobilised into dendritic spines feeding the increased need for receptors and membrane at the spine neck and head. In this review, we discuss the importance of endosomal-recycling and the role of key proteins which control these processes in the context of LTP.

Class I Rab11-family interacting proteins are binding targets for the Rab14 GTPase.

BACKGROUND INFORMATION: Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11-FIPs (family interacting proteins). This protein family has been sub-classified into two groups - class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11-interacting protein)] and class II FIPs (FIP3 and FIP4). RESULTS: In the present study we identify the class I FIPs as dual Rab-binding proteins by demonstrating that they also interact with Rab14 in a GTP-dependent manner. We show that these interactions are specific for the class I FIPs and that they occur via their C-terminal regions, which encompass the previously described RBD (Rab11-binding domain). Furthermore, we show that Rab14 significantly co-localizes with the TfnR (transferrin receptor) and that Rab14 Q70L co-localizes with Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase. Additionally, we show that during cytokinesis Rab14 localizes to the cleavage furrow/midbody. CONCLUSIONS: The data presented in the present study, which identifies the class I FIPs as the first putative effector proteins for the Rab14 GTPase, indicates greater complexity in the Rab-binding specificity of the class I FIP proteins.

Etiologies and predictors of diagnosis in nonresponsive celiac disease.

BACKGROUND & AIMS: Nonresponsive celiac disease (NRCD) is a common problem affecting from 7% to 30% of celiac patients. Because NRCD comprises varied and potentially morbid entities, efficient and cost-effective patient care requires knowledge of the specific causes of this disorder. The aim of this study was to determine the common etiologies of NRCD in a tertiary referral center. METHODS: All cases of biopsy examination-proven celiac disease (CD) seen at our institution over the preceding 5 years were included in this study. NRCD was defined as a failure to respond to at least 6 months of treatment with a gluten-free diet or the re-emergence of symptoms or laboratory abnormalities typical of CD while still on treatment with a gluten-free diet. RESULTS: A total of 113 patients with NRCD meeting the earlier-described criteria were seen from a total of 603 patients with CD (19%), however, among patients for whom we provided primary specialist care the incidence of NRCD was 10% (P < .001). Gluten exposure was the most common cause of NRCD (36%), followed by irritable bowel syndrome (22%), refractory CD (10%), lactose intolerance (8%), and microscopic colitis (6%). The mean immunoglobulin A tissue transglutaminase level in the gluten-exposed group was 67 vs 17 U/mL (normal, <20) for other diagnoses (P < .05). Weight loss and male sex were highly predictive of refractory CD (P < .05 and < .001, respectively). CONCLUSIONS: NRCD is a common phenomenon affecting 10%-19% of celiac patients. A limited number of etiologies account for the majority of cases. Clinical factors may be used to guide evaluation.

Current gut-directed therapies for irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder that can present with a wide array of symptoms that make treatment difficult. Current therapies are directed at relieving symptoms of abdominal pain or discomfort, bloating, constipation, and diarrhea. Pharmacologic agents used to treat IBS-associated pain include myorelaxants, peppermint oil, and peripherally acting opiates. Dicyclomine and hyoscyamine, the two myorelaxants available in the United States, have not been proven effective in reducing abdominal pain in patients with IBS. The efficacy of peppermint oil is debated, but methodological problems with existing studies preclude definitive judgment. Loperamide is ineffective for relief of abdominal pain. For IBS patients with excessive abdominal bloating, a small number of studies suggest that bacterial eradication with gut-directed antibiotics and bacterial reconstitution with nonpathogenic probiotics may reduce flatulence. For constipation-predominant (C-IBS) symptoms, current treatment options include fiber supplementation, polyethylene glycol, and tegaserod. Soluble fibers (ispaghula, calcium polycarbophil, psyllium) are more effective than insoluble fibers (wheat bran, corn fiber) in alleviating global symptoms and relieving constipation, although fiber in general has marginal benefit in treatment of overall IBS symptoms. Polyethylene glycol increases bowel frequency in chronic constipation, but its overall efficacy against IBS is unclear. Tegaserod, a 5-HT(4) agonist, demonstrates superiority over placebo in improving bowel frequency and stool consistency and alleviating abdominal pain and bloating in women with C-IBS. Overall global symptoms are modestly improved with tegaserod when compared with placebo. Additional agents under investigation for C-IBS include the ClC(2) chloride channel opener lubiprostone, mu-opioid receptor antagonist alvimopan, and 5-HT(4) agonist renzapride. For diarrhea-predominant (D-IBS) symptoms, available therapies include loperamide, alosetron, and clonidine. Alosetron, a 5-HT(3) antagonist, is superior to placebo for reducing bowel frequency, improving stool consistency, and relieving abdominal pain in women with D-IBS. However, alosetron is available under a restricted license because of concerns for ischemic colitis and severe constipation necessitating colectomy. Clonidine may be helpful in alleviating global symptoms for D-IBS patients.

Human antibody response to surface layer proteins in Clostridium difficile infection.

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Surface layer proteins (SLPs) are the most abundant surface localised proteins expressed by C. difficile. The aim of this study was to examine the humoral immune response to C. difficile SLPs and its potential role in protection from C. difficile associated diarrhoea (CDAD). Serum antibodies to SLPs from C. difficile were measured by ELISA in a cohort of 146 patients (55 patients with CDAD, 34 asymptomatic carriers, and 57 controls). No significant difference was detected in serum IgM, IgA or IgG antibody levels between cases, carriers or control groups at any of the time points tested. However, patients with recurrent episodes of C. difficile diarrhoea had significantly lower IgM-anti-SLP levels than patients with a single episode on days 1, 3, 6 and 9 (p = 0.05, p = 0.009, p = 0.02, p = 0.049). The adjusted odds ratio for recurrent diarrhoea associated with a low day 3 serum IgM anti-SLP antibody level was 24.5 (95% confidence interval; 1.6-376.3). Further studies which examine the specific anti-SLP antibody responses to the colonising strain are warranted to determine if immune responses to C. difficile SLPs play a role in protection from CDAD.

Clostridium difficile vaccine and serum immunoglobulin G antibody response to toxin A.

There is a strong association between serum antibody responses to toxin A and protection against Clostridium difficile diarrhea. A parenteral C. difficile toxoid vaccine induced very-high-level responses to anti-toxin A immunoglobulin G (IgG) in the sera of healthy volunteers. After vaccination, the concentrations of anti-toxin A IgG in the sera of all 30 recipients exceeded the concentrations that were associated with protection in previous clinical studies. Furthermore, the median concentration of serum anti-toxin A IgG in the test group was 50-fold higher than the previous threshold. These findings support the feasibility of using a vaccine to protect high-risk individuals against C. difficile-associated diarrhea and colitis.