Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta

The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.     

Diet-Induced Metabolic Improvements in a Hamster Model of Hypercholesterolemia Are Strongly Linked to Alterations of the Gut Microbiota

The mammalian gastrointestinal microbiota exerts a strong influence on host lipid and cholesterol metabolism. In this study, we have characterized the interplay among diet, gut microbial ecology, and cholesterol metabolism in a hamster model of hypercholesterolemia. Previous work in this model had shown that grain sorghum lipid extract (GSL) included in the diet significantly improved the high-density lipoprotein (HDL)/non-HDL cholesterol equilibrium (T. P. Carr, C. L. Weller, V. L. Schlegel, S. L. Cuppett, D. M. Guderian, Jr., and K. R. Johnson, J. Nutr. 135:2236-2240, 2005). Molecular analysis of the hamsters'' fecal bacterial populations by pyrosequencing of 16S rRNA tags, PCR-denaturing gradient gel electrophoresis, and Bifidobacterium-specific quantitative real-time PCR revealed that the improvements in cholesterol homeostasis induced through feeding the hamsters GSL were strongly associated with alterations of the gut microbiota. Bifidobacteria, which significantly increased in abundance in hamsters fed GSL, showed a strong positive association with HDL plasma cholesterol levels (r = 0.75; P = 0.001). The proportion of members of the family Coriobacteriaceae decreased when the hamsters were fed GSL and showed a high positive association with non-HDL plasma cholesterol levels (r = 0.84; P = 0.0002). These correlations were more significant than those between daily GSL intake and animal metabolic markers, implying that the dietary effects on host cholesterol metabolism are conferred, at least in part, through an effect on the gut microbiota. This study provides evidence that modulation of the gut microbiota-host metabolic interrelationship by dietary intervention has the potential to improve mammalian cholesterol homeostasis, which has relevance for cardiovascular health.The mammalian gut microbiota interacts intimately with its host, affecting both host metabolic and immunological phenotypes with important consequences for health (18, 22, 32). Recent studies have revealed complex linkages between the gut microbiome and host metabolism, with the microbes exerting effects on the energy balance by influencing glucose and lipid metabolism (2, 7, 28). This intimate metabolic relationship is most likely the consequence of a long coevolutionary process that resulted in a mutualistic relationship between the host and its microbial partners (25). However, life in industrialized societies has introduced profound changes into the human environment (e.g., diet, antibiotics, hospital deliveries, hygiene, etc.) that are markedly different from the conditions to which humans have evolved and that are likely to have occurred too abruptly for the human microbiome to adjust. Consequently, aberrations of the gut microbiota induced through lifestyle factors could be relevant to the etiology of several complex human diseases whose occurrence has markedly increased in developed countries. Interestingly, imbalances in the gut microbiota have been reported for obesity, type 1 and 2 diabetes, some allergies, and inflammatory bowel diseases in humans and animal models (7, 24, 43, 45, 48). The connection between gut bacteria and disease suggests an intriguing paradigm on how to view and potentially treat complex diseases. Specific bacterial populations in the intestine could be pharmaceutical targets to maintain or restore metabolic functions (6, 17).Coronary heart disease (CHD) continues to be a major cause of death in developed countries and is another example of a “western disease” that is less common in underdeveloped countries but increases in frequency with adoption of western customs (4). Most risk factors for CHD (obesity, high blood pressure, type 2 diabetes, heredity, high cholesterol, and diet) have been linked to the gut microbiota (7, 17, 20, 30, 45), and gut bacteria have been suggested to play a role in the etiology of cardiovascular disease (16, 33). Cholesterol metabolism is a key factor in susceptibility to CHD, and as early as 1959, it has been shown that germfree rats have higher serum cholesterol concentrations than their conventional counterparts do (12). Several mechanisms have been proposed by which gut bacteria could influence host cholesterol metabolism (13). Bacterial conversions of bile acids (such as the formation of secondary bile acids) are likely to play a role, as they affect enterohepatic circulation, de novo synthesis of bile acids, emulsification, and cholesterol absorption (10, 28, 30). A further mechanism by which gut bacteria might influence cholesterol metabolism is through Fiaf (fasting-induced adipocyte factor), which is selectively suppressed in the intestinal epithelium by the gut microbiota (1, 2). Fiaf is an important regulator of lipid metabolism (e.g., through its inhibition of lipoprotein lipase) and has been shown to increase total cholesterol and high-density lipoprotein (HDL) cholesterol levels when overexpressed in transgenic mice (26).There are several reasons why hamsters are an excellent model for studying the metabolic relationships among diet, cholesterol metabolism, and gut microbiota in relation to health. First, hamsters are omnivorous, and their blood lipid profiles respond to diets in a predictive manner similar to humans (5). Second, unlike mice and rats which lack cholesterol ester transfer protein, hamsters exhibit all of the enzymatic pathways in lipoprotein and bile metabolism that are also present in humans. They exhibit limited hepatic synthesis of cholesterol and bile acids, resulting in more relevant data when extrapolating to humans (23). Third, hamsters develop atherosclerosis in a predictive manner in response to dietary manipulation (31).Using the Golden Syrian hamster model, Carr and coworkers have shown that the hexane-extractable lipid fraction of grain sorghum whole kernels (GSL), when included in the hamsters'' diet, leads to a significant reduction of plasma non-HDL and liver cholesterol levels while increasing HDL cholesterol levels (8). We extended this research and performed a comprehensive molecular characterization of the fecal microbiota of the hamsters by pyrosequencing of 16S rRNA tags, denaturing gradient gel electrophoresis (DGGE), and Bifidobacterium specific quantitative real-time PCR (qRT-PCR) in order to test whether metabolic effects of GSL were associated with specific modifications of the gut microbiota.     

The effects of nitrate loading on the invasive macrophyte Hydrilla verticillata and two common, native macrophytes in Florida

Rising nitrate concentrations in the water column and the spread of invasive, non-native macrophytes are two major threats to Florida's oligotrophic, freshwater ecosystems. We used a replicated mesocosm experiment to test the effects of elevated nitrate concentrations in the water on the growth of the invasive macrophyte Hydrilla verticillata and two common, native submerged macrophytes Vallisneria americana and Sagittaria kurziana. Results from this study indicate that nitrate concentrations of 1.0 mg L⁻¹ NO₃-N in the water increased the final dry-weight biomass of H. verticillata by 2.75 times, while having no statistical effect on the growth of the two native species. Additionally, H. verticillata grew at a faster rate than the two native species in the low nitrate treatments accounting for 82% of the total biomass, indicating that it may have the capacity to invade relatively pristine communities. In waters where nitrate concentrations continue to rise, the cost of control efforts for H. verticillata may substantially increase in the future.     

GABA Increases Electrical Excitability in a Subset of Human Unmyelinated Peripheral Axons

Background

A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established.

Methodology/Principal Findings

Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1–100 µM) increased electrical excitability in a subset (ca. 40%) of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABA_A receptors, being mimicked by bath application of the GABA_A agonist muscimol (0.1–30 µM) while the GABA_B agonist baclofen (10–30 µM) was without effect. Increases in excitability produced by GABA (10–30 µM) were blocked by the GABA_A antagonists gabazine (10–20 µM), bicuculline (10–20 µM) and picrotoxin (10–20 µM).

Conclusions/Significance

Functional GABA_A receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABA_A receptor modulation may therefore regulate segmental and peripheral components of nociception.     

A systematic review of population based epidemiological studies in Myasthenia Gravis

Background  

The aim was to collate all myasthenia gravis (MG) epidemiological studies including AChR MG and MuSK MG specific studies. To synthesize data on incidence rate (IR), prevalence rate (PR) and mortality rate (MR) of the condition and investigate the influence of environmental and technical factors on any trends or variation observed.     

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (alpha and beta) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.     

Escherichia coli DnaA protein: specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.