p.R254Q mutation in the aquaporin‐2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin‐induced phosphorylation

Vasopressin regulates human water homeostasis by re‐distributing homotetrameric aquaporin‐2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP‐dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2‐p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2‐p.S256A, which mimics non‐phosphorylated AQP2. In polarized MDCK cells, AQP2‐p.R254Q was retained and was distributed similarly to that of unstimulated wt‐AQP2 or AQP2‐p.S256A. Upon co‐expression, AQP2‐p.R254Q interacted with, and retained wt‐AQP2 in intracellular vesicles. In contrast to wild‐type AQP2, forskolin did not increase AQP2‐p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2‐p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley‐Liss, Inc.     

A guide to performing difficult bimanual coordination tasks: just follow the yellow brick road

Both discrete and continuous bimanual coordination patterns are difficult to effectively perform when the two limbs are required to perform different movements patterns, move at different velocities and/or move different amplitudes unless some form of integrated feedback is provided. The purpose of the present experiment was to determine the degree to which a complex bimanual coordination pattern could be performed when integrated feedback and movement template are provided. The complex bimanual coordination pattern involved reciprocal movements of the two limbs under different difficulty requirements. As defined by Fitts’ index of difficulty (ID), the left arm (ID = 3, A = 16°, W = 4°) task was of lower difficulty than the right arm task (ID = 5, A = 32°, W = 2°). Note that the left and right limb movements are also different in terms of movement time, movement velocity, accuracy requirements and amplitude as well as one movement was continuous and the other intermittent. Participants were provided 2 blocks of 9 trials in the bimanual condition (30 s/trial). Following the bimanual phase, participants performed two unimanual test trials—one with each limb. The results demonstrated that the performance for each limb in the bimanual condition was similar to the performance for the same limb and conditions in the unimanual control conditions. The similarity was indicated by the same movement speed, movement structure, endpoint variability and hit rates for the bimanual and unimanual conditions. The results support our hypothesis that people can overcome the intrinsic difficulties associated with performing complex bimanual coordination patterns when provided appropriate perceptual information feedback that allows them to detect and correct coordination errors.     

The role of auditory and visual models in the production of bimanual tapping patterns

An experiment was designed to determine the effectiveness of auditory and visual models in the learning of a 2:3 bimanual tapping pattern. Participants were randomly assigned to an auditory model, visual model, auditory + visual model, or a control (visual metronome) group. The task for all groups was to tap a left side force transducer with the left hand and a right side force transducer with the right hand in attempt to produce the desired 2:3 bimanual coordination pattern. The auditory model consisted of a series of tones representing the goal pattern played prior to each practice trial. The visual model consisted of a visual display representing the goal tapping pattern. Visual pacing metronomes were provided to the control group. The right and left side metronomes flashed during the trial in a pattern representing the goal tapping pattern. Subjects in all groups performed 14 practice trials consisting of 15 s each devoted to tapping the goal pattern (total practice time = 3.5 min). A retention test without the aid of the models or metronomes was administered following the practice trials. The results for the model groups indicated extremely effective performance of the bimanual coordination patterns for the auditory, visual, and auditory + visual model conditions with not only the relative, but also the absolute characteristics of the models exhibited during retention testing. Retention performance for the visual metronome condition was less accurate and more variable than the three model conditions. In addition, the auditory + visual model condition resulted in retention performance that was more stable than the auditory model condition.     

CD40 expression by gingival fibroblasts: correlation of phenotype with function

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non- hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine- secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up- regulates secretion of these cytokines in vitro.      

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

A Mouse Model of Lethal Synergism Between Influenza Virus and Haemophilus influenzae

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.Infections with influenza virus cause mild to severe respiratory illness and may result in death in vulnerable human populations.^1,2,3,4 On average, influenza causes three to five million cases of severe illness per year worldwide and over 200,000 hospitalizations and 36,000 deaths in the United States alone.¹ 5 to 20% of the US population are infected annually. While healthy adults typically experience only acute uncomplicated infection, influenza virus predisposes the lungs to bacterial co-infections,^5,6,7 which cause significant additional morbidity, particularly in young children, elderly and immunocompromised individuals.^8,9,10,11,12 Secondary bacterial infections may also significantly increase mortality in the population as a whole during influenza pandemics.^13,14,15,16 For example, in the 1918 influenza pandemic, which killed approximately 50 million people worldwide, while infection with the virus alone could be lethal, the majority of deaths appeared to result from secondary bacterial pneumonia.^16,17,18,19 The most common bacterial agents mediating such secondary infections in the U.S. are Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae.^20,21,22,23H. influenzae is a small Gram-negative coccobacillus that exists in capsulated or non-capsulated forms. H. influenzae is a common cause of otitis media, acute sinusitis, bronchitis, pneumonia and exacerbations of chronic obstructive pulmonary disease.^{24,25,26,27,28,29} A vaccine against H. influenzae type b (Hib) has greatly reduced the incidence of invasive disease, such as meningitis, caused by this organism in children under 5 years of age.^30,31,32,33 However, Hib invasive disease in children remains a problem in countries where the vaccine is not widely available.^32,34,35 Furthermore, other encapsulated and non-typable (NTHi) forms are increasing in frequency as causes of illness in young children.^28,29,36 During the 1918 influenza pandemic, H. influenzae was often isolated from the autopsied lungs of young adults, a subpopulation who do not usually die from influenza infection.¹⁶Early studies by Shope³⁷ showed that infection of pigs with both influenza virus and H. influenzae suis resulted in severe disease or death, whereas the individual agents induced only mild infection. Similarly, Orticoni et al³⁸ reported that administration of both filtrates of nasal secretions from 1918 influenza patients and H. influenzae caused a lethal disease in guinea pigs, but there was no effect if either agent was administered alone. Influenza also increases the susceptibility of new-born rats to H. influenzae-induced meningitis³⁹ and synergizes with the bacteria in the development of otitis media in the chinchilla.⁴⁰ A single study conducted in 1945 showed that infection with both influenza virus and H. influenzae killed mice at doses that were sublethal when either agent was administered alone.⁴¹ However, this study pre-dated modern immunological techniques, precluding assessment of the underlying mechanism.To investigate the pathobiological mechanisms further, we established a model of influenza and H. influenzae co-infection in mice. Herein, we report that H. influenzae synergizes with influenza virus to cause more severe disease in immunocompetent adult mice, leading to 100% lethality at doses that cause no mortality when the agents are give individually. The mechanism leading to disease exacerbation does not involve T or B cells, and thus appears to be mediated by innate immunity. However, tumor necrosis factor (TNF), interleukin-6 (IL-6), and Toll-like receptor (TLR)-4 are not essential for synergistic lethality in this model.     

Balancing high accrual and ethical recruitment in paediatric oncology: a qualitative study of the 'look and feel' of clinical trial discussions

Background  

High accrual to clinical trials enables new treatment strategies to be tested rapidly, accurately and with generalisability. Ethical standards also must be high so that participation is voluntary and informed. However, this can be difficult to achieve in trials with complex designs and in those which are closely embedded in clinical practice. Optimal recruitment requires a balance of both ethical and accrual considerations. In the context of a trial of stratified treatments for children with acute lymphoblastic leukaemia (UKALL2003) we examined how recruitment looked to an observer and how it felt to the parents, to identify how doctors' communication could promote or inhibit optimal recruitment.     

An analysis of correspondence between unique rabies virus variants and divergent big brown bat (Eptesicus fuscus) mitochondrial DNA lineages

The literature supports that unique rabies virus (RABV) variants are often compartmentalized in different species of bats. In Colorado, two divergent mtDNA lineages of big brown bats (Eptesicus fuscus) co-occur. RABV associated with this species also segregates into two clades. We hypothesized that unique RABV variants might be associated with mtDNA lineages of Colorado big brown bats. DNA was extracted from brain tissue of rabid big brown bats, the ND2 gene was amplified to determine mtDNA lineage, and the lineage was compared to a previously derived phylogenetic analysis of the RABV N gene. No correspondence was found between host bat lineage and RABV variant.     

Tissue-engineered follicles produce live, fertile offspring

Oocytes grown in vitro are of low quality and yield few live births, thus limiting the ability to store or bank the ova of women wishing to preserve their fertility. We applied tissue engineering principles to the culture of immature mouse follicles by designing an alginate hydrogel matrix to maintain the oocyte's 3- dimensional (3D) architecture and cell-cell interactions in vitro. A 3D culture mimics the in vivo follicle environment, and hydrogel-encapsulated follicles develop mature oocytes within the capacity for fertilization similar to that of oocytes matured in vivo. Embryos derived from cultured oocytes fertilized in vitro and transferred to pseudopregnant female mice were viable, and both male and female offspring were fertile. Our results demonstrate that alginate hydrogel-based 3D in vitro culture of follicles permits normal growth and development of follicles and oocytes. This system creates new opportunities for discovery in follicle biology and establishes a core technology for human egg banks for preservation of fertility.