Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+]i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.
Store-operated calcium channels (SOCs) are a nearly ubiquitous Ca2+ entry pathway stimulated by numerous cell surface receptors via the reduction of Ca2+ concentration in the ER. The discovery of STIM proteins as ER Ca2+ sensors and Orai proteins as structural components of the Ca2+ release-activated Ca2+ (CRAC) channel, a prototypic SOC, opened the floodgates for exploring the molecular mechanism of this pathway and its functions. This review focuses on recent advances made possible by the use of STIM and Orai as molecular tools. I will describe our current understanding of the store-operated Ca2+ entry mechanism and its emerging roles in physiology and disease, areas of uncertainty in which further progress is needed, and recent findings that are opening new directions for research in this rapidly growing field.
Store-operated Ca2+ channels (SOCs) in the plasma membrane are activated by reduced ER luminal Ca2+ concentrations. This involves the rearrangement of STIM (Ca2+ sensor) and Orai proteins (calcium channel components) in the cell.
Patients with vestibular dysfunction have visual, perceptual, and
postural deficits. While there is considerable evidence that a semicircular
canal prosthesis that senses angular head velocity and stimulates canal
ampullary nerves can improve vision by augmenting the vestibulo-ocular reflex,
no information is available regarding the potential utility of a canal
prosthesis to improve perceptual deficits. In this study we investigated the
possibility that electrical stimulation of canal afferents could be used to
modify percepts of head orientation. Two rhesus monkeys were trained to align a
light bar parallel to gravity, and were tested in the presence and absence of
electrical stimulation provided by an electrode implanted in the right posterior
canal. While the monkeys aligned the light bar close to the true earth-vertical
without stimulation, when the right posterior canal was stimulated their
responses deviated towards their left ear, consistent with a misperception of
head tilt towards the right. The deviation of the light bar from the
earth-vertical exceeded the torsional deviation of the eyes, indicating that the
perceptual changes were not simply visual in origin. Eye movements recorded
during electrical stimulation in the dark were consistent with isolated
activation of right posterior canal afferents, with no evidence of otolith
stimulation. These results demonstrate that electrical stimulation of canal
afferents affects the perception of head orientation, and therefore suggest that
motion-modulated stimulation of canal afferents by a vestibular prosthesis could
potentially improve vestibular percepts in patients lacking normal vestibular
To evaluate cataract risk in eyes of patients with AIDS and cytomegalovirus (CMV) retinitis and to identify risk factors.
Prospective cohort study.
Patients with AIDS and CMV retinitis.
Patients 13 years of age and older were enrolled between 1998 and 2008. Demographic and clinical characteristics, slit-lamp biomicroscopy findings, and dilated ophthalmoscopy results were documented at quarterly visits. Cataract status was determined at the initial visit (prevalence) and at follow-up visits (incidence).
Main Outcome Measures
For cataract, a high grade of lens opacity by biomicroscopy to which best-corrected visual acuity worse than 20/40 was attributed. Eyes that had undergone cataract surgery before enrollment or between visits also were counted as having cataract.
Seven hundred twenty-nine eyes of 489 patients diagnosed with CMV retinitis were evaluated. Higher prevalence was observed for patients with bilateral versus unilateral CMV retinitis (adjusted odds ratio [aOR], 2.74; 95% confidence interval [CI], 1.76–4.26) and, among unilateral CMV retinitis cases, for eyes with retinitis versus without retinitis (15% vs. 1.4%; P<0.0001). The age-adjusted prevalence of cataract among CMV retinitis cases was higher than that in a population-based sample (P<0.0001). Cataract prevalence increased with age (aOR, 11.77; 95% CI, 2.28–60.65 for age ≥60 years vs. younger than 40 years) and longer duration of retinitis (aOR, 1.36; 95% CI, 1.20–1.54 per year). Among eyes with CMV retinitis initially free of cataract, the cataract incidence was 8.1%/eye-year (95% CI, 6.7%–10.0%). Prior retinal detachment was associated with higher cataract risk (if repaired with silicone oil: adjusted hazard ratio [aHR], 10.37; 95% CI, 6.51–16.52; otherwise: aHR, 2.90; 95% CI, 1.73–4.87). Large CMV retinitis lesions also were associated with higher risk of cataract (for involvement of 25–49% retinal area: aHR, 2.30; 95% CI, 1.51–3.50; for ≥50% involvement: aHR, 3.63; 95% CI, 2.18–6.04), each with respect to ≤24% involvement, as were anterior segment inflammation (aHR, 2.27; 95% CI, 1.59–3.25) and contralateral cataract (aHR, 2.52; 95% CI, 1.74–3.66).
Cytomegalovirus retinitis is associated with a high absolute and relative risk of cataract. Among several risk factors, large retinal lesion size and use of silicone oil in retinal detachment repair are potentially modifiable, albeit not in all cases. Cataract is likely to be an increasingly important cause of visual morbidity in this population.
vestibular; migraine; vertigo; psychophysics; thresholds
Antagonists of N-type voltage-gated calcium channels (VGCC), Cav2.2, can manage severe chronic pain with intrathecal use and may be effective systemically. A series of novel ω-conotoxins that selectively inhibit N-type VGCCs was isolated from Conus catus. In the present study, the potency and reversibility of ω-conotoxins CVID, CVIE and CVIF to inhibit N-type calcium currents were investigated in mouse isolated dorsal root ganglion (DRG) neurons. The systemic potency of each ω-conotoxin to reverse signs of mouse chronic inflammatory pain was also compared.
In DRG neurons, the rank order of potency to inhibit N-type calcium currents was CVIE > CVIF > CVID. After subcutaneous administration, CVID and CVIE, but not CVIF, partially reversed impaired weight bearing in mice injected with Freund’s complete adjuvant (CFA) three days prior to testing. No side-effects associated with systemic administration of ω-conotoxins were observed.
The present study indicates a potential for CVID and CVIE to be developed as systemically active analgesics with no accompanying neurological side-effects.
Calcium channel; Conotoxin; Inflammatory pain
Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining.
Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies.
Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.
ConoSorter; Cone snail; Venomics; Transcriptome; Proteome; Conopeptides; Conotoxins
Vestibular symptoms caused by migraine, referred to as vestibular migraine, are a frequently diagnosed but poorly understood entity. Based on recent evidence that normal subjects generate vestibular-mediated percepts of head motion and reflexive eye movements using different mechanisms, we hypothesized that percepts of head motion may be abnormal in vestibular migraine. We therefore measured motion detection thresholds in patients with vestibular migraine, migraine patients with no history of vestibular symptoms, and normal subjects using the following paradigms: roll rotation while supine (dynamically activating the semicircular canals); quasi-static roll tilt (statically activating the otolith organs); and dynamic roll tilt (dynamically activating the canals and otoliths). Thresholds were determined while patients were asymptomatic using a staircase paradigm, whereby the peak acceleration of the motion was decreased or increased based on correct or incorrect reports of movement direction. We found a dramatic reduction in motion thresholds in vestibular migraine compared to normal and migraine subjects in the dynamic roll tilt paradigm, but normal thresholds in the roll rotation and quasi-static roll tilt paradigms. These results suggest that patients with vestibular migraine may have enhanced perceptual sensitivity (e.g. increased signal-to-noise ratio) for head motions that dynamically modulate canal and otolith inputs together.
Vestibular; migraine; vertigo; psychophysics; thresholds
The clinical features of mitochondrial disease are complex and highly variable, leading to challenges in establishing a specific diagnosis. Despite being one of the most commonly occurring inherited genetic diseases with an incidence of 1/5000, ~90% of these complex patients remain without a DNA-based diagnosis. We report our efforts to identify the pathogenetic cause for a patient with typical features of mitochondrial disease including infantile cataracts, CPEO, ptosis, progressive distal muscle weakness, and ataxia who carried a diagnosis of mitochondrial disease for over a decade.
Whole exome sequencing and bioinformatic analysis of these data were conducted on the proband.
Exome sequencing studies showed a homozygous splice site mutation in SETX, which is known to cause Spinocerebellar Ataxia, Autosomal Recessive 1 (SCAR1). Additionally a missense mutation was identified in a highly conserved position of the OCRL gene, which causes Lowe Syndrome and Dent Disease 2.
This patient’s complex phenotype reflects a complex genetic etiology in which no single gene explained the complete clinical presentation. These genetic studies reveal that this patient does not have mitochondrial disease but rather a genocopy caused by more than one mutant locus. This study demonstrates the benefit of exome sequencing in providing molecular diagnosis to individuals with complex clinical presentations.
Encephalomyopathy; Lowe syndrome; OCRL; SETX; Diagnostics; Genocopy
Mutations in MPZ cause CMT1B, the second most frequent cause of CMT1. Elegant studies with Ser63del mice suggest that Ser63del MPZ is retained in the ER where it activates the unfolded protein response (UPR) that contributes to the neuropathy. Clinical information about patients with this mutation is limited. We present clinical and electrophysiological data on a large multigenerational family with CMT1B caused by Ser63del MPZ. The patients have a classical CMT1 phenotype that is much less severe than that of patients with Arg98Cys MPZ that also activates the UPR. These results suggest that clinical presentation along cannot predict which MPZ mutations will be retained in the ER and activate the UPR.
We determined the fraction of families in a well-characterized cohort with a provisional diagnosis of autosomal dominant retinitis pigmentosa (adRP) that have disease-causing mutations in the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene or the retinitis pigmentosa 2 (RP2) gene.
Families with a provisional clinical diagnosis of adRP, and a pedigree consistent with adRP but no male-to-male transmission were selected from a cohort of 258 families, and tested for mutations in the RPGR and RP2 genes with di-deoxy sequencing. To facilitate testing of RPGR in “adRP” families that had no male members available for testing, the repetitive and purine-rich ORF15 of RPGR was subcloned and sequenced in heterozygous female subjects from 16 unrelated families.
Direct sequencing of RPGR and RP2 allowed for identification of a disease-causing mutation in 21 families. Of these “adRP” families 19 had RPGR mutations, and two had RP2 mutations. Subcloning and sequencing of ORF15 of RPGR in female subjects identified one additional RPGR mutation. Of the 22 mutations identified, 15 have been reported previously.
These data show that 8.5% (22 in 258) of families thought to have adRP truly have X-linked retinitis pigmentosa (XLRP). These results have substantive implications for calculation of recurrence risk, genetic counseling, and potential treatment options, and illustrate the importance of screening families with a provisional diagnosis of autosomal inheritance and no male-to-male transmission for mutations in X-linked genes. Mutations in RPGR are one of the most common causes of all forms of retinitis pigmentosa.
This study identifies the fraction of families in a well-characterized cohort with a provisional diagnosis of autosomal dominant retinitis pigmentosa (adRP) that have disease-causing mutations in the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene or the retinitis pigmentosa 2 (RP2) gene.
Bacterial biofilms are becoming a significant societal problem: biofilms form dental plaque, coat ships causing biofouling, and cling onto medical instruments and implants. Understanding how these surface-bound communities are formed is crucial for the development of suitable strategies for their dispersal. At the heart of a switch that commits Bacilli and related species to form biofilms is a transcriptional regulator called SinR and its multiple antagonists. In this addendum, we discuss an alternative model to account for how one of the antagonists is regulated by controlled proteolysis.
Bacillus; SinR; adaptive responses; biofilm; protein interactions; transcriptional repression
Short stature and later maturation of youth artistic gymnasts are often attributed to the effects of intensive training from a young age. Given limitations of available data, inadequate specification of training, failure to consider other factors affecting growth and maturation, and failure to address epidemiological criteria for causality, it has not been possible thus far to establish cause–effect relationships between training and the growth and maturation of young artistic gymnasts. In response to this ongoing debate, the Scientific Commission of the International Gymnastics Federation (FIG) convened a committee to review the current literature and address four questions: (1) Is there a negative effect of training on attained adult stature? (2) Is there a negative effect of training on growth of body segments? (3) Does training attenuate pubertal growth and maturation, specifically, the rate of growth and/or the timing and tempo of maturation? (4) Does training negatively influence the endocrine system, specifically hormones related to growth and pubertal maturation? The basic information for the review was derived from the active involvement of committee members in research on normal variation and clinical aspects of growth and maturation, and on the growth and maturation of artistic gymnasts and other youth athletes. The committee was thus thoroughly familiar with the literature on growth and maturation in general and of gymnasts and young athletes. Relevant data were more available for females than males. Youth who persisted in the sport were a highly select sample, who tended to be shorter for chronological age but who had appropriate weight-for-height. Data for secondary sex characteristics, skeletal age and age at peak height velocity indicated later maturation, but the maturity status of gymnasts overlapped the normal range of variability observed in the general population. Gymnasts as a group demonstrated a pattern of growth and maturation similar to that observed among short-, normal-, late-maturing individuals who were not athletes. Evidence for endocrine changes in gymnasts was inadequate for inferences relative to potential training effects. Allowing for noted limitations, the following conclusions were deemed acceptable: (1) Adult height or near adult height of female and male artistic gymnasts is not compromised by intensive gymnastics training. (2) Gymnastics training does not appear to attenuate growth of upper (sitting height) or lower (legs) body segment lengths. (3) Gymnastics training does not appear to attenuate pubertal growth and maturation, neither rate of growth nor the timing and tempo of the growth spurt. (4) Available data are inadequate to address the issue of intensive gymnastics training and alterations within the endocrine system.
Electronic supplementary material
The online version of this article (doi:10.1007/s40279-013-0058-5) contains supplementary material, which is available to authorized users.
To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.
Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA.
We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region).
This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.
Identification of mutations in 15% of the screened patients has important implications for guiding clinicians who are ordering genetic testing and provides a strong argument for screening the RPGR gene in simplex cases of retinal degenerative diseases.
Aicardi syndrome is a rare X-linked disorder that has been characterized classically by agenesis of the corpus callosum, seizures, and the finding of chorioretinal lacunae. This triad has been augmented more recently by central nervous system and ocular findings. The goal of this study is to determine how frequently other ophthalmologic findings are associated with Aicardi syndrome.
A single ophthalmologist recorded the ocular and adnexal findings of 40 girls with this disorder at the annual meeting of an Aicardi syndrome family support group. For each subject, the examiner performed facial anthropometrics, portable biomicroscopy, and, where feasible, indirect ophthalmoscopy.
The most common findings were chorioretinal lacunae in 66 (88%) of 75 eyes and optic nerve abnormalities in 61 (81%) of 75 eyes. Other less common findings included persistent pupillary membrane in 4 (5%) of 79 eyes and anterior synechiae in 1 of 79 eyes (1%).
Although the ophthalmic hallmark and defining feature of Aicardi syndrome is the cluster of distinctive chorioretinal lacunae surrounding the optic nerve(s), the spectrum of ocular, papillary, and retinal anomalies varies widely, from nearly normal to dysplasia of the optic nerve and to severe microphthalmos.
RbpA is a small non–DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA–σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.
Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous retinal dystrophy. The causes of LCA have been unraveled partially at the molecular level. At least 14 genes have been reported that, when mutated, result in LCA. To understand the roles of the known genes in LCA, a group of outbred subjects from 60 apparently either recessive families, with one or more affected individuals, or isolated patients were evaluated. One affected individual from each family underwent comprehensive mutational analysis by direct DNA sequencing of all coding regions and splice junctions of 13 LCA genes. Mutations were identified in 70% of individuals. CEP290 made the largest contribution to the identified mutations, providing 43% of those mutant alleles. We identified seven families in which affected individuals with two mutant alleles, sufficient to cause disease, had an additional mutation at a second LCA locus. Our findings suggest that mutational load can be important to penetrance of the LCA phenotype.
The expression, purification and crystallization of the trans-acting acyltransferase PksC from the bacillaene hybrid polyketide synthase/nonribosomal peptide synthetase is described. The crystals belonged to the orthorhombic space group P212121 and diffracted to 1.44 Å resolution.
The antibiotic bacillaene is biosynthesized in Bacillus subtilis by a hybrid type 1 modular polyketide synthase/nonribosomal peptide synthetase of the trans-acyltransferase (trans-AT) class. Within this system, the essential acyl-group loading activity is provided by the action of three free-standing trans-acting acyltransferases. Here, the recombinant expression, purification and crystallization of the bacillaene synthase trans-acting acyltransferase PksC are reported. A diffraction data set has been collected from a single PksC crystal to 1.44 Å resolution and the crystal was found to belong to the orthorhombic space group P212121.
trans-acyltransferases; polyketide synthases; bacillaene
To assess the contributions of the vestibular system to whole-body motion discrimination in the dark, we measured direction-recognition thresholds as a function of frequency for yaw rotation, superior-inferior translation (“z-translation”), inter-aural translation (“y-translation”), and roll-tilt for 14 normal subjects and for three patients following total bilateral vestibular ablation. The patients had significantly higher average threshold measurements than normal (p<0.01) for yaw-rotation (depending upon frequency, 5.4× to 15.7× greater), z-translation (8.3× to 56.8× greater), y-translation (1.7× to 4.5× greater), and roll tilt (1.3× to 3.0× greater) – establishing the predominant contributions of the vestibular system for these motions in the dark.
Semicircular Canals; Otolith Organs; Human; Thresholds
SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1–3, β1, β3, β4, γ1, γ4–5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.
We are studying the effectiveness of a semicircular canal prosthesis to improve postural control, perception of spatial orientation, and the VOR in rhesus monkeys with bilateral vestibular hypofunction. Balance is examined by measuring spontaneous sway of the body during quiet stance and postural responses evoked by head turns and rotation of the support surface; perception is measured with a task derived from the subjective visual vertical (SVV) test during static and dynamic rotation in the roll plane; and the angular VOR is measured during rotation about the roll, pitch, and yaw axes. After the normal responses are characterized, bilateral vestibular loss is induced with intratympanic gentamicin, and then multisite stimulating electrodes are chronically implanted into the ampullae of all three canals in one ear. The postural, perceptual, and VOR responses are then characterized in the ablated state, and then bilateral, chronic electrical stimulation is applied to the ampullary nerves using a prosthesis that senses angular head velocity in three-dimensions and uses this information to modulate the rate of current pulses provided by the implanted electrodes. We are currently characterizing two normal monkeys with these paradigms, and vestibular ablation and electrode implantation are planned for the near future. In one prior rhesus monkey tested with this approach, we found that a one-dimensional (posterior canal) prosthesis improved balance during head turns, perceived head orientation during roll tilts, and the VOR in the plane of the instrumented canal. We therefore predict that the more complete information provided by a three-dimensional prosthesis that modulates activity in bilaterally-paired canals will exceed the benefits provided by the one-dimensional, unilateral approach used in our preliminary studies.
The objective of this project was to investigate the relationships between total and regional distribution of body fat and tissue lutein (L) and zeaxanthin (Z) status. Healthy men and women (N = 100; average age: 22.5 year, average BMI: 23.4 kg/m2) were evaluated. Total body and regional fat mass were assessed by dual-energy X-ray absorptiometry (Hologic Delphi A). Serum LZ was measured using reverse phase high-performance liquid chromatography, and retinal LZ (referred to as macular pigment optical density; MPOD) was measured using heterochromatic flicker photometry. Body fat percentage (total and regional) was inversely related to MPOD (p < 0.01) but no significant relationship was found for serum LZ. Higher body fat percentage, even within relatively healthy limits, is associated with lower tissue LZ status. The results indicate that adiposity may affect the nutritional state of the retina. Such links may be one of the reasons that obesity promotes age-related degenerative conditions of the retina.
lutein; zeaxanthin; body fat; adiposity
The stressosome is a bacterial signalling complex that responds to environmental changes by initiating a protein partner switching cascade, which leads to the release of the alternative sigma factor, σB. Stress perception increases the phosphorylation of the stressosome sensor protein, RsbR, and the scaffold protein, RsbS, by the protein kinase, RsbT. Subsequent dissociation of RsbT from the stressosome activates the σB cascade. However, the sequence of physical events that occur in the stressosome during signal transduction is insufficiently understood.
Here, we use computational modelling to correlate the structure of the stressosome with the efficiency of the phosphorylation reactions that occur upon activation by stress. In our model, the phosphorylation of any stressosome protein is dependent upon its nearest neighbours and their phosphorylation status. We compare different hypotheses about stressosome activation and find that only the model representing the allosteric activation of the kinase RsbT, by phosphorylated RsbR, qualitatively reproduces the experimental data.
Our simulations and the associated analysis of published data support the following hypotheses: (i) a simple Boolean model is capable of reproducing stressosome dynamics, (ii) different stressors induce identical stressosome activation patterns, and we also confirm that (i) phosphorylated RsbR activates RsbT, and (ii) the main purpose of RsbX is to dephosphorylate RsbS-P.
Bacillus subtilis; Stressosome; Signalling; Cellular automaton; Stress response
The planar cell polarity (PCP) signaling pathway governs collective cell movements duringvertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly ofcilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.
Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen.