Aim. To report the aetiological spectrum and susceptibility patterns of bacteria isolated from patients with corneal ulceration. Method. The microbiological data of all patients with suspected infectious corneal ulceration who presented to the ocular microbiology service at this centre between 2005 and 2012 were reviewed retrospectively. Result. Microorganisms were recovered from 1665 (77%) of the 2170 ulcers. Bacterial isolates accounted for 1205 of the organisms isolated. The most common bacterial pathogens isolated were various species of Staphylococcus, representing 777 (64.5%), followed by Staphylococcus spp. (148; 12.3%) and Pseudomonas aeruginosa (117; 9.7%). High percentages of Gram-positive bacteria were susceptible to gatifloxacin (>94%), followed by ofloxacin and moxifloxacin. Almost 90% of Pseudomonas aeruginosa isolates were susceptible to ciprofloxacin and moxifloxacin. Sixty-two (44%) of 140 isolates of Streptococcus pneumoniae, 79 (14.8%) of 534 isolates of Staphylococcus epidermidis, and 33 (14%) of 234 isolates of Staphylococcus aureus were resistant to three or more antibiotics. Conclusion. Staphylococcus spp. were the most common bacterial pathogens isolated from patients with keratitis in this setting. High percentages of Gram-positive and Gram-negative bacteria were susceptible to gatifloxacin and moxifloxacin, respectively. Interestingly, a high percentage of Streptococcus pneumoniae isolates were found to be resistant to three or more antibiotics.
The angiosperm radiation has been linked to sharp declines in gymnosperm diversity and the virtual elimination of conifers from the tropics. The conifer family Podocarpaceae stands as an exception with highest species diversity in wet equatorial forests. It has been hypothesized that efficient light harvesting by the highly flattened leaves of several podocarp genera facilitates persistence with canopy-forming angiosperms, and the angiosperm ecological radiation may have preferentially favoured the diversification of these lineages. To test these ideas, we develop a molecular phylogeny for Podocarpaceae using Bayesian-relaxed clock methods incorporating fossil time constraints. We find several independent origins of flattened foliage types, and that these lineages have diversified predominantly through the Cenozoic and therefore among canopy-forming angiosperms. The onset of sustained foliage flattening podocarp diversification is coincident with a declining diversification rate of scale/needle-leaved lineages and also with ecological and climatic transformations linked to angiosperm foliar evolution. We demonstrate that climatic range evolution is contingent on the underlying state for leaf morphology. Taken together, our findings imply that as angiosperms came to dominate most terrestrial ecosystems, competitive interactions at the foliar level have profoundly shaped podocarp geography and as a consequence, rates of lineage diversification.
Podocarpaceae; molecular phylogeny; divergence time estimates; plant evolution
Misfolding of ΔF508 CFTR underlies pathology in most CF patients. F508 resides in the first nucleotide binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the ΔF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores ΔF508 maturation and function to wild type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.
The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/solubility were identified. Genes with profound effects in the solubility assay were selected for validation by independent biochemical assays. Six of 10 validated genes had a significant effect on SOD1 solubility and folding in a SOD1 promoter-driven β-gal assay, indicating that global screening of cellular targets using such protein solubility/folding assay is viable and can be adapted for other misfolding diseases.
cDNA expression cloning screen; amyotrophic lateral sclerosis; superoxide dismutase 1; protein solubility assay
ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5′-(β,γ-imino)triphosphate and adenosine 5′-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction.
It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.
The proteasome plays a central role in the degradation of regulatory and misfolded proteins. Current models suggest that substrates access the internal catalytic sites by processively threading their termini through the gated substrate channel. Here, we found that latent (closed) and activated (open) proteasomes degraded two natively disordered substrates at internal peptide bonds even when they lacked accessible termini, suggesting that these substrates themselves promoted gating of the proteasome. This endoproteolysis provides a molecular mechanism for regulated release of transcription factors from inactive precursors as well as a means of accessing internal folding defects of misfolded multidomain proteins.
Inefficient folding of CFTR into a functional three-dimensional structure is the basic pathophysiologic mechanism leading to most cases of cystic fibrosis. Knowledge of the structure of CFTR and placement of these mutations into a structural context would provide information key for developing targeted therapeutic approaches for cystic fibrosis. As a large polytopic membrane protein containing disordered regions, intact CFTR has been refractory to efforts to solve a high-resolution structure using X-ray crystallography. The following chapters summarize current efforts to circumvent these obstacles by utilizing NMR, electron microscopy, and computational methodologies and by development of experimental models of the relevant domains of CFTR.
CFTR structure; NMR; EM; crystallography; spectroscopy
Despite the availability of several studies to clarify taxonomic problems on the highly threatened yews of the Hindu Kush-Himalaya (HKH) and adjacent regions, the total number of species and their exact distribution ranges remains controversial. We explored the use of comprehensive sets of morphological, molecular and climatic data to clarify taxonomy and distributions of yews in this region.
A total of 743 samples from 46 populations of wild yew and 47 representative herbarium specimens were analyzed. Principle component analyses on 27 morphological characters and 15 bioclimatic variables plus altitude and maximum parsimony analysis on molecular ITS and trnL-F sequences indicated the existence of three distinct species occurring in different ecological (climatic) and altitudinal gradients along the HKH and adjacent regions Taxus contorta from eastern Afghanistan to the eastern end of Central Nepal, T. wallichiana from the western end of Central Nepal to Northwest China, and the first report of the South China low to mid-elevation species T. mairei in Nepal, Bhutan, Northeast India, Myanmar and South Vietnam.
The detailed sampling and combination of different data sets allowed us to identify three clearly delineated species and their precise distribution ranges in the HKH and adjacent regions, which showed no overlap or no distinct hybrid zone. This might be due to differences in the ecological (climatic) requirements of the species. The analyses further provided the selection of diagnostic morphological characters for the identification of yews occurring in the HKH and adjacent regions. Our work demonstrates that extensive sampling combined with the analysis of diverse data sets can reliably address the taxonomy of morphologically challenging plant taxa.
Cystic fibrosis is a lethal genetic disease caused by lack of functional cystic fibrosis transmembrane conductance regulator (CFTR) proteins at the apical surface of secretory epithelia. CFTR is a multidomain protein, containing five domains, and its functional structure is attained in a hierarchical folding process. Most CF-causing mutations in CFTR, including the most common mutation, a deletion of phenylalanine at position 508 (ΔF508), are unable to properly fold into this functional native three dimensional structure. Currently, no high-resolution structural information about full length CFTR exists. However, insight has been gained through examining homologous ABC transporter structures, molecular modeling, and high-resolution structures of individual, isolated CFTR domains. Taken together, these studies indicate that the prevalent ΔF508 mutation disrupts two essential steps during the development of the native structure: folding of the first nucleotide binding domain (NBD1) and its later association with the fourth intracellular loop (ICL4) in the second transmembrane domain (TMD2). Therapeutics to rescue ΔF508 and other mutants in CFTR can be targeted to correct defects that occur during the complex folding process. This article reviews the structural relationships between CFTR and ABC transporters and current knowledge about how CFTR attains its structure–with a focus on how this process is altered by CF-causing mutations in a manner targetable by therapeutics.
CFTR; cystic fibrosis; ABC transporter; membrane protein structure; multidomain protein folding
Amyotrophic Lateral Sclerosis (ALS) is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1) that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43), a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis.
Glutathione has a wide range of functions; it is an endogenous anti-oxidant and plays a key role in the maintenance of intracellular redox balance and detoxification of xenobiotics. Several studies have indicated that children with autism spectrum disorders may have altered glutathione metabolism which could play a key role in the condition.
A systematic literature review and meta-analysis was conducted of studies examining metabolites, interventions and/or genes of the glutathione metabolism pathways i.e. the γ-glutamyl cycle and trans-sulphuration pathway in autism spectrum disorders.
Thirty nine studies were included in the review comprising an in vitro study, thirty two metabolite and/or co-factor studies, six intervention studies and six studies with genetic data as well as eight studies examining enzyme activity.
The review found evidence for the involvement of the γ-glutamyl cycle and trans-sulphuration pathway in autistic disorder is sufficiently consistent, particularly with respect to the glutathione redox ratio, to warrant further investigation to determine the significance in relation to clinical outcomes. Large, well designed intervention studies that link metabolites, cofactors and genes of the γ-glutamyl cycle and trans-sulphuration pathway with objective behavioural outcomes in children with autism spectrum disorders are required. Future risk factor analysis should include consideration of multiple nutritional status and metabolite biomarkers of pathways linked with the γ-glutamyl cycle and the interaction of genotype in relation to these factors.
γ-glutamyl cycle; Trans-sulphuration pathway; Metabolites; Genes; Supplementation; Autism spectrum disorders
Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO4) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO4 and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.
Zinc; Cytotoxicity; DNA damage; Genomic stability; Human oral keratinocytes; Micronuclei
There are distinct molecular pathologies for two cystic fibrosis–causing mutations in the first transmembrane span of the CF transmembrane conductance regulator protein. These results have implications for understanding the mechanisms of membrane protein integration and folding and the effects of disease-causing mutants.
Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.
Aim. To describe keratitis due to Chaetomium sp. occurring in a 65-year-old woman who presented with a corneal ulcer with hypopyon of the right eye with a history of trauma by vegetable matter. Method. Multiple scrapings were obtained from the ulcer. A lactophenol cotton blue wet mount and a Gram-stained smear of the scrapings were made. Scrapings were also inoculated onto various culture media. Results. Direct microscopy of corneal scrapings revealed moderate numbers of septate fungal hyphae. Greenish-yellow-coloured fungal colonies with aerial mycelium were observed in culture of the corneal scrapes. On the basis of colony characteristics and conidial structure, the fungal isolate was identified as Chaetomium sp. The patient was treated with topical natamycin (5%) hourly and cyclopentolate 1% drops 3 times a day. After 4 weeks of therapy, the hypopyon had disappeared, the epithelial defect had healed, and the stromal infiltration had almost completely resolved; the visual acuity of the eye improved from hand movements to (1/2)/60. Conclusion. Fungi of the genus Chaetomium, which are rare causes of human disease (systemic mycosis, endocarditis, subcutaneous lesions), may also cause ocular lesions.
The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR’s first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure–function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.
Protein folding; protein degradation; antibody generation; cell surface protein detection; research consortium; www.cftrfolding.org
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane – thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.
CFTR structure; CFTR folding; stability; NBD1; spectroscopy
A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. α-Synuclein (αSyn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves αSyn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length αSyn. Three point mutations in αSyn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of αSyn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of αSyn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype αSyn. These truncations accelerated fibrillization of all α-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of αSyn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of αSyn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated α-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.
20S proteasome; Endoproteolysis; Parkinson disease; αSynuclein; Amyloid; Soluble oligomers; Cytotoxicity; Liposomes
To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin.
In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9–14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca2+-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR.
Significantly lower mean activities of Ca2+-ATPase and Na+/K+ -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca2+-ATPase and Na+/K+-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools.
The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.
Clinical tests have shown that the dynamics of a human arm, controlled using Functional Electrical Stimulation (FES), can vary significantly between and during trials. In this paper, we study the application of Reinforcement Learning to create a controller that can adapt to these changing dynamics of a human arm. Development and tests were done in simulation using a two-dimensional arm model and Hill-based muscle dynamics. An actor-critic architecture is used with artificial neural networks for both the actor and the critic. We begin by training it using a Proportional Derivative (PD) controller as a supervisor. We then make clinically relevant changes to the dynamics of the arm and test the actor-critic’s ability to adapt without supervision in a reasonable number of episodes.
Functional Electrical Stimulation; Motor Control; Reinforcement Learning; Actor-Critic; Function Approximation
Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1)1-3. Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB)4, gephyrin (GPHN)5 and RhoGEF collybistin (ARHGEF9)6. However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes2-7. Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5)8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na+ binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na+/Cl−-dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where defects in postsynaptic receptors have been identified, similar symptoms could result from defects in the cognate presynaptic neurotransmitter transporter.
A drama documentary examines the story of a mentally ill junior doctor who rejects conventional treatment but is determined to stay in medicine. Philip Thomas reviews
Fluid and HCO3– secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO3– in secretory glands is fueled by Na+/HCO3– cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl–/HCO3– exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP3) receptor–binding protein released with IP3 (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO3– secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase–regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.