Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.     

Morphology and ecological significance of intra-annual radial cracks in living conifers

 Intra-annual radial cracks were studied on 294 cross-sections of Norway spruce sampled at two forest sites in the eastern Alps (Italy) and from seven isolated trees in the Jura region (Switzerland). Cracks were occasionally accompanied by traumatic resin canals in the wood that was formed after the cracking. Most of the cracks, however, were without such canals. Traumatic resin canals are not significantly more abundant in tree rings formed after cracking, and their occurrence is not related to the cracking. Cracks developed when the cambium was inactive. Water imbalances during the early spring, due to transpiration losses and inadequate moisture supply from very cold roots, are the likely cause of these cracks. Received: 21 February 1996 / Accepted: 14 June 1996     

Novel and Unique Diagnostic Biomarkers for Bacillus anthracis Infection

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10³ to 10⁵ CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10³ CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease which is initiated, in its most severe form, by inhalation of spores. Due to the severity of the disease, the ease of respiratory infection, and the extreme resistance of the spores to unfavorable environmental conditions, B. anthracis is considered a potential biological warfare agent (for a review, see references 8, 10, 35, 56, and 62), and in recent years, the need for novel reliable diagnostic approaches, improved vaccination strategies, novel therapeutic targets, and a better understanding of the pathogenesis of anthrax has been widely acknowledged.Inhaled B. anthracis spores are taken up by alveolar macrophages and germinate into vegetative bacilli which eventually invade the bloodstream, where they multiply massively and secrete toxins and virulence factors. Anthrax is toxinogenic in the sense that the bacterial binary exotoxin is necessary for the onset of the disease (54), yet other factors may be required for the colonization and expansion of bacteria in the host (15, 18, 31, 32, 37, 46, 65, 66, 70, 83). The toxin is composed of the following three proteins: protective antigen (PA), which mediates binding to the receptor on target cells and internalization of the toxin components (14, 74); lethal factor, a zinc protease targeting several mitogen-activated protein kinases (52); and edema factor (EF), a calmodulin-dependent adenylate cyclase (55, 57). The genes encoding the three exotoxin components are located on the native virulence plasmid pXO1. Genes encoding proteins with functions involved in the synthesis of the second major B. anthracis virulence determinant, an immunologically inert polyglutamyl capsule that protects bacteria from phagocytosis, are located on a second native virulence plasmid, pXO2 (56).In humans, the initial symptoms of inhalation anthrax are nonspecific and reminiscent of influenza-like upper respiratory tract infections. The second stage is characterized by high fever, respiratory failure, dyspnea, and shock. Unless patients are treated promptly, death occurs within 24 h of the onset of the second stage, preceded by massive bacteremia (27, 34, 73, 76). The mandatory treatment for anthrax is based on administration of antibiotics (17, 76), yet study of the disease in animal models has clearly established that the time of antibiotic administration postinfection is crucial for the effectiveness of the treatment, strongly supporting the importance of rapid diagnosis (2, 47, 48). At present, due to the severity of the disease and its rapid progression, treatment is administered to each person with confirmed contact with contaminated areas (76).Early accurate diagnosis of anthrax is complicated by the rarity of the disease and the nonspecific nature of the symptoms. Microbiologic identification of anthrax is based on the nonhemolytic nature of the typically white-gray colonies and the rod morphology of the gram-positive nonmotile bacilli detected in clinical samples or blood cultures (16, 19, 30, 73, 78). Immunofluorescence and immunohistochemistry targeted to bacterial proteins are not routinely conducted. Later in the course of the disease, seroconversion in response to the various components of the toxin may serve only as a retrospective confirmation of initial exposure. With the advent of genetic methodologies, B. anthracis in cultures inoculated with clinical and forensic samples can be detected specifically and accurately by PCR, usually designed to amplify genes located on the native virulence plasmids (30). Recently, the use of PA as a disease biomarker was suggested, owing to the presence of this protein in detectable amounts in the circulation of infected animals (53, 71). EF and lethal factor can be detected in the circulation only at later stages of infection (30).In recent years, the search for novel biomarkers of disease, including bacterial infections, has exploited the approach of global biological inspection based on functional genomic or proteomic studies (64, 85). We previously documented such global surveys, combined with a serological study of B. anthracis (5, 6, 20, 21, 22, 38, 39), for identification of vaccine and diagnostic marker candidates among extracellular (secreted or membranal) proteins. These studies indeed revealed a list of proteins that can serve as potential biomarkers, based on their immunogenicity (which probes their in vivo expression), abundance under various culture conditions, and functional relatedness to infection. In the present study, the search was extended by directly addressing the presence of bacterial secreted proteins in the circulation of B. anthracis-infected rabbits, using specific antibodies generated by DNA vaccination against the previously selected immunogenic proteins. Visualization of bacterial proteins in the circulation of infected animals was achieved only following depletion of highly abundant serum proteins by an affinity chromatography protocol. The search enabled the successful detection, in addition to PA, of three secreted proteins uniquely expressed by B. anthracis, i.e., HtrA (BA3660), the BA1952 endopeptidase, and a protein of unknown function (BA0796). All of these proteins are potential virulence-related factors. This is the first communication of the presence of B. anthracis secreted proteins other than the bacterial toxin in the circulation of infected animals, and their identification strongly supports the validity of the reductional screening approach for selection of disease biomarkers.     

Intra- and inter-item associations doubly dissociate the electrophysiological correlates of familiarity and recollection

Single-process models of recognition memory posit that recognizing is based on a unidimensional value of global memory strength. By contrast, dual-process models propose the existence of two independent processes subserving the explicit recognition of previously encountered episodes, namely "familiarity" and "recollection." Familiarity represents a noncontextual form of recognition that may only support the retrieval of associative information when the to-be-associated information can be unitized, such as when two photographs depicting the same person are memorized (intra-item associations). Conversely, recollection enables retrieving associations between arbitrarily linked information, such as associations between photographs of different persons (inter-item associations). By measuring event-related brain potentials (ERPs), we obtained a double dissociation of familiarity and recollection that strongly favors dual-process accounts of recognition memory: the electrophysiological correlate of familiarity was significantly larger for intra- than for inter-item associations. Conversely, the electrophysiological correlate of recollection was significantly larger for inter- than for intra-item associations.     

Using short columns to speed up LC–MS quantification in MS binding assays

The present study describes the use of short columns to speed up LC–MS quantification in MS binding assays. The concept of MS binding assays follows closely the principle of traditional radioligand binding but uses MS for the quantification of bound marker thus eliminating the need for a radiolabelled ligand. The general strategy of increasing the throughput of this type of binding assay by the use of short columns is exemplified for NO 711 binding addressing GAT1, the most prevalent GABA transporter in the CNS. Employing short RP-18 columns with the dimension of 20 mm × 2 mm and 10 mm × 2 mm at flow rates up to 1000 μL/min in an isocratic mode retention times of 8–9 s and chromatographic cycle times of 18 s could be achieved. Based on the internal standard [²H₁₀]NO 711 fast chromatography methods were developed for four different columns that enabled quantification of NO 711 in a range from 50 pM up to 5 nM directly out of reconstituted matrix samples without further sample preparation. A validation of the established methods with respect to linearity, intra- and inter-batch accuracy and precision showed that the requirements according to the FDA guideline for bioanalytical methods are met. Furthermore the established short column methods were applied to the quantification of NO 711 in saturation experiments. The results obtained (i.e., K_d- and B_max-values) were almost identical as compared to those determined employing standard column dimension (55 mm × 2 mm).