The effect of human rhinovirus-16 on thermo-TRP channels, hypotonic activation and ATP release

Human rhinovirus (RV-16) infections are major cause of upper respiratory tract infections (URTI) and are associated with acute exacerbations of asthma and COPD. URTIs are associated with excessive production of mucus within the airway, particularly in sufferers of chronic respiratory conditions. This mucus may lead to altered osmolarity within the airway and contribute to hypotonic stimulation of the airway epithelium and airway hypersensitivity leading to cough. Hypotonic stimulation has previously been shown to activate transient receptor potential (TRP) vanilloid 4 (TRPV4) ultimately leading to ATP release via pannexin-1. RV-16 has been previously shown to cause upregulation of TRP channel expression in the airway, however, the mechanism of common cold cough is unknown. We hypothesise that infection with RV-16 causes increased ATP release in the airway leading to airway hypersensitivity and increased cough frequency.Infection of airway epithelial (A549) and transfected astrocytoma (1321N1) cell lines with synthetic viral stimulus poly(I:C) failed to cause any change in expression or function of TRPA1, V1, V4 or P2X3 when monitored using RT-PCR, western blot analysis or intracellular calcium signalling. However, infection with RV-16 led to reduced function of thermo-TRP channels without concurrent reduction in expression although function was restored to basal levels by late infection.The role of hypotonic stimulation on A549 cells was investigated with the aim of further delineating the mechanism of ATP release following hypotonic stimulation. Calcium-calmodulin kinase was identified to play an integral role in downstream ATP release, and that blockade of calcium-calmodulin kinase or pannexin-1 significantly reduced ATP release following hypotonic stimulation. Furthermore, immunofluorescent analysis identified a distinct distribution pattern of pannexin-1 prior to and post hypotonic stimulation, which could be effectively abolished through inhibition of pannexin-1, or significantly reduced following inhibition of calcium-calmodulin kinase, TRPV4 or MLC phosphorylation.Finally, RV-16 infection led to increased ATP release from 72 hours through 168 hours post infection, independent of further stimulus at a time point which coincides with the development of viral cough in vivo. Furthermore, RV-16 infection led to a glycolytic shift in cell metabolism without any increase in cell death.In summary, RV-16 induced ATP release, mediated by calcium-calmodulin kinase, and through TRPV4 and pannexin-1 activation, may underpin the development of a viral cough during an upper respiratory tract infection

Genetic Analysis of TON2 Dependent Processes in Microtubule Organization in Arabidopsis thaliana

Microtubules carry out many functions within the cell. They\u27re used during mitosis and meiosis to move chromosomes to opposite sides of the cell so that cell division can occur. They\u27re also used as structural support for the cell and are involved in establishing the cell shape. Microtubules are also able to reorient from a longitudinal orientation to a transverse orientation in response to gibberellic acid and auxin (two hormones that are involved in cell elongation). The mechanism for how this reorientation occurs is unknown. The TONNEAU2 (TON2) protein is necessary for proper microtubule organization. We looked at how TON2 affects microtubule orientation and nucleation in response to the hormones gibberellic acid and auxin and found that while the ton2 mutant can reorient in response to the hormones it is less efficient. We also found that nucleation mode is not causing the reorientation. Previously it was found that TON2 interacts with the POK protein. POK is a protein that interacts with the phragmoplast and helps orient it for proper formation of the cell plate. We looked at POK localization and timing in ton2 mutants, which lack a PPB. POK\u27s presence overlapped with the cell cycle stages that TON2 is present during and localized to the same place the PPB used to inhabit. RIC1 is another putative TON2 interacting protein. We tested the genetic relationship between RIC1 and TON2 in cell shape determination. Our data shows that TON2 could be epistatic to RIC1 by comparing the neck and lobe lengths of pavement cells in wild type, ric1-1 mutants, ton2 mutants, and ric1-1 ton2 double mutants, but more data is needed to confirm this hypothesis. The double mutant had the same phenotype of no or few and small lobes as the ton2 mutant

Rhinovirus-16 increases ATP release in A549 cells without concomitant increase in production

Human rhinovirus (RV) is the most common cause of upper respiratory tract infection (URTI) and chronic airway disease exacerbation. Cough is present in 50–80% of URTI cases, accompanied by heightened airway hypersensitivity, yet no effective treatment currently exists for this infectious cough. The mechanism by which RV causes cough and airway hypersensitivity in URTI is still unknown despite recent advances in potential therapies for chronic cough.The effect of RV-16 infection (MOI 1) on intracellular ATP stores and ATP release in A549 alveolar epithelial cells was measured.RV-16 infection was found to significantly increase (by 50% from basal at 24 h) followed by decrease (by 50% from basal at 48 and 72 h) intracellular ATP concentrations, while increasing ATP release (from 72 h) independently of secondary stimulation. This effect was mimicked by intercellular adhesion molecule 1 receptor binding alone through ultraviolet-inactivated sham control. In addition, RV-16-infected cells became more sensitive to secondary stimulation with both hypotonic and isotonic solutions, suggestive of a hypersensitive response. These responses were not mediated via increased TRPV4 or pannexin-1 whole-cell expression as determined by Western blotting. Interestingly, the increased ATP release seen was not a result of increased mitochondrial ATP production.Thus, this is the first report demonstrating that RV-16 infection of airway epithelial cells causes hypersensitivity by increasing ATP release. These finding provide a novel insight into the process by which viruses may cause cough and identify a potential target for treatment of viral and post-viral cough

A Pilot Study on the Effects of Curcumin on Parasites, Inflammation, and Opportunistic Bacteria in Riding Horses

Twelve riding horses were utilized to examine the effects of curcumin on intestinal parasites, inflammation, and the fecal shedding of Streptococcus bovis/equinus complex (SBEC), Clostridium difficile and Clostridium perfringens. Known for having anti-inflammatory, antimicrobial, and antiparasitic properties it was hypothesized that curcumin would decrease parasite shedding, inflammation, and opportunistic bacteria found in the GIT of riding horses. Horses were randomly assigned to one of the following treatments (n = 6/treatment): 1) no curcumin, control (CON); or 2) 15 g of 95% pure curcumin, (CUR). Curcumin was dosed per day for 30 d. Fecal samples were evaluated for shedding of ova and concentrations of selected bacteria. Blood samples taken pre and post riding intervals and evaluated for erythrocyte sedimentation rate (ESR) for inflammation. All data were analyzed for repeated measures. Treatment had no effect (P ≥ 0.58) on total fecal egg count, strongyles, or ascarids. Treatment had no effect on ESR (P ≤ 0.42); however, ESR decreased (P = 0.0006) on d 14 in CUR horses. Treatment had no effect (P ≥ 0.34) on concentrations of SBEC, C. difficile, or C. perfringens. Curcumin was not an effective compound against intestinal parasites or fecal microbial strains examined when administered for 30 days; but could potentially decrease inflammation. Curcumin has been observed to have many beneficial effects in other species, however, more research is needed to evaluate those benefits in horses

The complement system and human autoimmune diseases

Genetic deficiencies of early components of the classical complement activation pathway (especially C1q, r, s, and C4) are the strongest monogenic causal factors for the prototypic autoimmune disease systemic lupus erythematosus (SLE), but their prevalence is extremely rare. In contrast, isotype genetic deficiency of C4A and acquired deficiency of C1q by autoantibodies are frequent among patients with SLE. Here we review the genetic basis of complement deficiencies in autoimmune disease, discuss the complex genetic diversity seen in complement C4 and its association with autoimmune disease, provide guidance as to when clinicians should suspect and test for complement deficiencies, and outline the current understanding of the mechanisms relating complement deficiencies to autoimmunity. We focus primarily on SLE, as the role of complement in SLE is well-established, but will also discuss other informative diseases such as inflammatory arthritis and myositis

A database of whole-body action videos for the study of action, emotion, and untrustworthiness

We present a database of high-definition (HD) videos for the study of traits inferred from whole-body actions. Twenty-nine actors (19 female) were filmed performing different actions—walking, picking up a box, putting down a box, jumping, sitting down, and standing and acting—while conveying different traits, including four emotions (anger, fear, happiness, sadness), untrustworthiness, and neutral, where no specific trait was conveyed. For the actions conveying the four emotions and untrustworthiness, the actions were filmed multiple times, with the actor conveying the traits with different levels of intensity. In total, we made 2,783 action videos (in both two-dimensional and three-dimensional format), each lasting 7 s with a frame rate of 50 fps. All videos were filmed in a green-screen studio in order to isolate the action information from all contextual detail and to provide a flexible stimulus set for future use. In order to validate the traits conveyed by each action, we asked participants to rate each of the actions corresponding to the trait that the actor portrayed in the two-dimensional videos. To provide a useful database of stimuli of multiple actions conveying multiple traits, each video name contains information on the gender of the actor, the action executed, the trait conveyed, and the rating of its perceived intensity. All videos can be downloaded free at the following address: http://www-users.york.ac.uk/~neb506/databases.html. We discuss potential uses for the database in the analysis of the perception of whole-body actions

Thiocillin contributes to the ecological fitness of Bacillus cereus ATCC 14579 during interspecies interactions with Myxococcus xanthus

The soil-dwelling delta-proteobacterium Myxococcus xanthus is a model organism to study predation and competition. M. xanthus preys on a broad range of bacteria mediated by lytic enzymes, exopolysaccharides, Type-IV pilus-based motility, and specialized metabolites. Competition between M. xanthus and prey bacterial strains with various specialized metabolite profiles indicates a range of fitness, suggesting that specialized metabolites contribute to prey survival. To expand our understanding of how specialized metabolites affect predator–prey dynamics, we assessed interspecies interactions between M. xanthus and two strains of Bacillus cereus. While strain ATCC 14579 resisted predation, strain T was found to be highly sensitive to M. xanthus predation. The interaction between B. cereus ATCC 14579 and M. xanthus appears to be competitive, resulting in population loss for both predator and prey. Genome analysis revealed that ATCC 14579 belongs to a clade that possesses the biosynthetic gene cluster for production of thiocillins, whereas B. cereus strain T lacks those genes. Further, purified thiocillin protects B. cereus strains unable to produce this specialized metabolite, strengthening the finding that thiocillin protects against predation and contributes to the ecological fitness of B. cereus ATCC 14579. Lastly, strains that produce thiocillin appear to confer some level of protection to their own antibiotic by encoding an additional copy of the L11 ribosomal protein, a known target for thiopeptides. This work highlights the importance of specialized metabolites affecting predator–prey dynamics in soil microenvironments