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1.  Inefficient SRP Interaction with a Nascent Chain Triggers a mRNA Quality Control Pathway 
Cell  2014;156(0):146-157.
Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a novel mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2) and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain, and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).
PMCID: PMC3931426  PMID: 24439374
2.  Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors 
Neuropharmacology  2015;88:63-73.
GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology.
For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors.
Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators.
This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.
•Neurosteroid potentiation at GABAA receptors is modulated by protein kinase activity.•Synaptic-type α1β3γ2L receptor potentiation is up or down-regulated by PKC activity.•PKC-mediated modulation occurs via β3 subunit S408 & S409 phosphorylation.•Potentiation at extrasynaptic-type α4β3δ receptors is additionally regulated by α4S443.•Neurosteroids facilitate phosphorylation of GABAA receptors via βS408,S409.
PMCID: PMC4239298  PMID: 25278033
Neurosteroid; Protein kinase; Synaptic GABAA receptors; Extrasynaptic GABAA receptors; Phosphorylation; THDOC, tetrahydro-deoxycorticosterone; BIM-I, bisindolylmaleimide I; PMA, phorbol-12-myristate-13-acetate; THIP, 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol
3.  Chronic exertional compartment syndrome of the forearm in elite rowers: a technique for mini-open fasciotomy and a report of six cases 
Hand (New York, N.Y.)  2013;8(4):450-453.
Chronic exertional compartment syndrome (CECS) of the forearm may occur in sports requiring prolonged grip strength. CECS is a function of increasing pressure following muscle expansion within an inelastic tissue envelope resulting in compromise of perfusion and tissue function. Typical symptoms are pain, distal paraesthesia and loss of function. The condition is self-limiting and resolves completely between periods of activity. With no effective medical treatment, the gold standard remains four compartment open fasciotomy (Söderberg, J Bone Joint Surg Br 78(5):780–2, 1996; Wasilewski and Asdourian, Am J Sports Med 19(6):665–7, 1991). Minimally invasive techniques have been described (Croutzet et al., Tech Hand Up Extrem Surg 13(3):137–40, 2009) but have a risk of neuro-vascular injury, especially to the ulnar nerve while releasing the deep flexor compartment. We present a safe technique used with six elite rowers for mini-open fasciotomy to minimise scarring and time away from training while reducing the risk of neurovascular injury.
PMCID: PMC3840767  PMID: 24426965
Rowing; Muscle injury and inflammation; Chronic exertional compartment syndrome
4.  Evolutionary Diversification of New Caledonian Araucaria 
PLoS ONE  2014;9(10):e110308.
New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a ‘museum’ for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.
PMCID: PMC4207703  PMID: 25340350
5.  Evolutionary dynamics of emblematic Araucaria species (Araucariaceae) in New Caledonia: nuclear and chloroplast markers suggest recent diversification, introgression, and a tight link between genetics and geography within species 
BMC Evolutionary Biology  2014;14(1):171.
New Caledonia harbours a highly diverse and endemic flora, and 13 (out of the 19 worldwide) species of Araucaria are endemic to this territory. Their phylogenetic relationships remain largely unresolved. Using nuclear microsatellites and chloroplast DNA sequencing, we focused on five closely related Araucaria species to investigate among-species relationships and the distribution of within-species genetic diversity across New Caledonia.
The species could be clearly distinguished here, except A. montana and A. laubenfelsii that were not differentiated and, at most, form a genetic cline. Given their apparent morphological and ecological similarity, we suggested that these two species may be considered as a single evolutionary unit. We observed cases of nuclear admixture and incongruence between nuclear and chloroplast data, probably explained by introgression and shared ancestral polymorphism. Ancient hybridization was evidenced between A. biramulata and A. laubenfelsii in Mt Do, and is strongly suspected between A. biramulata and A. rulei in Mt Tonta. In both cases, extensive asymmetrical backcrossing eliminated the influence of one parent in the nuclear DNA composition. Shared ancestral polymorphism was also observed for cpDNA, suggesting that species diverged recently, have large effective sizes and/or that cpDNA experienced slow rates of molecular evolution. Within-species genetic structure was pronounced, probably because of low gene flow and significant inbreeding, and appeared clearly influenced by geography. This may be due to survival in distinct refugia during Quaternary climatic oscillations.
The study species probably diverged recently and/or are characterized by a slow rate of cpDNA sequence evolution, and introgression is strongly suspected. Within-species genetic structure is tightly linked with geography. We underline the conservation implications of our results, and highlight several perspectives.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0171-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4182765  PMID: 25189104
Admixture; Closely related species; Conifers; Diversification; Hotspot; Hybridization; Introgression; Phylogeography; Population genetics; Systematics
6.  ‘A reservoir within a reservoir’ – An unusual complication associated with a defunctioned inflatable penile prosthesis reservoir 
Inflatable penile prostheses (IPP) have been a successful method of treating men with erectile dysfunction since the early 1970s. IPP are comprised of two intracorporal cylinders, a scrotal pump and a fluid reservoir.
We present a case of a retained reservoir in a sixty eight year old gentlemen presenting with a cystic abdominal mass and bothersome LUTS, 15 years after the removal of the penile components of a three-piece penile prosthesis. Percutaneous drainage of the cyst was performed, with four litres of purulent fluid evacuated. A midline laparotomy was required to remove the reservoir and drain the collection completely.
Inflammatory reaction and subsequent erosion of an IPP reservoir is an infrequent but severe complication of IPP insertion, replacement or infection. Infection remains the primary indication for penile prosthesis removal and in this setting removal of the reservoir is routine. A thorough literature search has identified that in the non-infective setting, the routine removal of the original reservoir is not standard practice during three-component IPP replacement. In patients with a history of IPP presenting with new LUTS, reservoir erosion should be considered in the differential diagnosis and investigation with cystoscopy and computed tomography included early in the investigatory armament of the urologist.
It is our belief that a defunctionalized reservoir serves no purpose; rather it can only cause trouble in the future. Consequently, at our institution we do not leave defunctionalized reservoirs in situ.
PMCID: PMC4189072  PMID: 25247874
Erectile dysfunction; Inflatable penile prosthesis; Lower urinary tract symptoms; Prosthesis reservoir
7.  Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress 
Mutagenesis  2013;28(4):475-484.
Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally susceptible to DNA damage.
PMCID: PMC3681539  PMID: 23766106
Nature communications  2013;4:10.1038/ncomms3816.
Alcohol-dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN × C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.
PMCID: PMC3843143  PMID: 24281383
10.  Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening 
Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ∼tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.
PMCID: PMC4010227  PMID: 20976528
Cystic fibrosis; CFTR; Structure-based virtual screening; Correctors; Potentiators; Modulators; F508; Docking; Homology modeling; YFP; Ussing chamber; Chemical chaperones; Pharmacological chaperones
11.  Scleral- fixated intraocular lens implantation in microspherophakia 
Indian Journal of Ophthalmology  2014;62(5):596-600.
In microspherophakia, abnormal laxity of the lenticular zonules leads to development of a spherical lens and possible subluxation. We evaluated long-term results of lens removal with scleral-fixated intraocular lens (SFIOL) implantation in microspherophakia.
Materials and Methods:
Case series. SF IOLs were implanted in four consecutive patients with bilateral microspherophakia (eight eyes [three with pupillary block and secondary glaucoma who underwent immediate surgery and five with only subluxation who underwent elective surgery]). Post-operative best-corrected visual acuity (BCVA), intraocular pressure (IOP) and lens position were evaluated periodically from day 1 to 18 months.
All patients were females (mean age 28 ± 7.03 years). In group 1 eyes (three eyes that presented with pupillary block), the mean BCVA improved from 0.008 decimals (preoperative) to 0.50 decimals (final post-operative visit); in group 2 eyes (the other five eyes), the mean BCVA improved from 0.12 ± 0.21 decimals to 0.73 ± 0.14 decimals. The preoperative mean IOP (54.53 ± 7.33 mmHg) in group 1 eyes was significantly (P = 0.03) higher than that (16 ± 4.30 mm Hg) in group 2 eyes. At final post-operative visit, the mean IOP (11.67 ± 2.88 mmHg) in group 1 eyes was not significantly different from that in group 2 eyes (13.0 ± 3.08 mmHg). All SFIOLs were well- centred at the final visit. None of the patients encountered any peroperative or postoperative complications.
SFIOLs may be an option for surgical management of microspherophakia.
PMCID: PMC4065512  PMID: 24881608
Ectopia lentis; microspherophakia; pupillary block; Scleral-fixated intraocular lenses
12.  Keratoconjunctivitis caused by an unusual retained conjunctival foreign body: A frequently unrecognized entity 
Indian Journal of Ophthalmology  2014;62(5):633-635.
The purpose of this study is to describe a case series of keratoconjunctivitis caused by a retained bindi (dot) in six children who presented to a tertiary eye care facility in Southern India.
Patients and Methods:
Over a period of 11 years (January 2000 and January 2012), six children (all female, ranging in age from 6 months to 3 years) were diagnosed with ocular manifestations subsequent to a retained bindi.
All patients presented with redness, photophobia, extensive lacrimation, and blepharospasm. Two patients presented with mucopurulent conjunctivitis, three patients with suppurative keratitis and one patient presented with corneal epithelial defect. After removal of the foreign body the response to topical antibiotics was good in five of six cases, whereas one patient required therapeutic keratoplasty.
Young children presenting with unilateral keratitis and conjunctivitis should alert the clinician to the possibility of a retained foreign body in the eye.
PMCID: PMC4065521  PMID: 24881616
Bindi; keratitis; keratoconjunctivitis
13.  Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways  
BioMed Research International  2014;2014:728019.
Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.
PMCID: PMC4020542  PMID: 24868545
14.  Paralogous Radiations of PIN Proteins with Multiple Origins of Noncanonical PIN Structure 
Molecular Biology and Evolution  2014;31(8):2042-2060.
The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.
PMCID: PMC4104312  PMID: 24758777
auxin; auxin transport; PIN protein; plant evolution; phylogeny; protein structure
15.  Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene 
Nature genetics  2013;45(10):10.1038/ng.2745.
Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation to clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 cystic fibrosis patients in registries and clinics in North America and Europe. Among these patients, 159 CFTR variants had an allele frequency of ≥0.01%. These variants were evaluated for both clinical severity and functional consequence with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of cystic fibrosis patients enabled assignment of 12 of the remaining 32 variants as neutral while the other 20 variants remained indeterminate. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically-relevant genomic variation.
PMCID: PMC3874936  PMID: 23974870
16.  ATP Binding and ATP Hydrolysis Play Distinct Roles in the Function of 26S Proteasome 
Molecular cell  2006;24(1):39-50.
The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.
PMCID: PMC3951175  PMID: 17018291
17.  Aberrant CFTR-dependent HCO3- transport inmutations associated with cystic fibrosis 
Nature  2001;410(6824):94-97.
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl− conductance in the sweat duct was discovered to be impaired in CF1, a finding that has been extended to all CFTR-expressing cells2–4. Subsequent cloning of the gene5,6 showed that CFTR functions as a cyclic-AMP-regulated Cl− channel7; and some CF-causing mutations inhibit CFTR Cl− channel activity2–4,8. The identification of additional CF-causing mutants with normal Cl− channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters3,4, including Cl−-coupled HCO3- transport9,10. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues11, indicating the importance of this activity. HCO3- and pH affect mucin viscosity12,13 and bacterial binding14,15. We have examined Cl−-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl− channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.
PMCID: PMC3943212  PMID: 11242048
18.  Gating of CFTR by the STAS domain of SLC26 transporters 
Nature cell biology  2004;6(4):343-350.
Chloride absorption and bicarbonate secretion are vital functions of epithelia1–6, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR7, which activates electrogenic chloride-bicarbonate exchange by SLC26T8. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.
PMCID: PMC3943213  PMID: 15048129
19.  CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices 
The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.
PMCID: PMC3943242  PMID: 17660831
20.  Weight loss on a structured hypocaloric diet with or without exercise improves emotional distress and quality of life in overweight and obese patients with type 2 diabetes 
To evaluate the effects of a caloric restricted weight loss program with or without supervised resistance exercise training (EX) on diabetes‐related emotional distress and quality of life (QOL) in overweight and obese patients with type 2 diabetes.
Materials and Methods
In a parallel design, 106 men and women with type 2 diabetes were randomized to a prescriptive 16‐week caloric restricted diet (D; 6,000–7,000 kJ/day), with (n = 65) or without (n = 41) EX (three times per week). Bodyweight, glycated hemoglobin, diabetes‐specific emotional distress (Problem Areas in Diabetes [PAID] questionnaire) and QOL (Diabetes‐39 [D‐39] questionnaire) was assessed pre‐ and post‐intervention.
A total of 84 participants completed the study (D n = 33, D + EX n = 51). Weight loss was significantly greater in D + EX compared with D (−11.4 ± 5.8 vs −8.8 ± 5.8 kg, P = 0.04 time × diet). Overall, there were significant improvements in glycated hemoglobin, PAID total score and the D‐39 dimensions of ‘diabetes control’, ‘anxiety and worry’, ‘sexual functioning’, ‘energy and mobility’, ‘overall rating of QOL’ and ‘severity of diabetes’ (P ≤ 0.01 for time). The D‐39 dimension, ‘social burden’, did not change (P = 0.07 for time). There was no difference between groups in the response for any of these variables (P ≥ 0.10).
A structured caloric restricted diet with or without EX improves emotional distress and QOL in overweight and obese patients with type 2 diabetes. This trial was registered with Australian New Zealand Clinical Trials Registry (; ACTR No: ACTRN12608000206325).
PMCID: PMC4025227  PMID: 24843744
Lifestyle intervention; Well‐being; Caloric restriction
21.  Antihypercholesterolemic and Antioxidative Potential of an Extract of the Plant, Piper betle, and Its Active Constituent, Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in Experimental Rats 
Hypercholesterolemia is a dominant risk factor for atherosclerosis and cardiovascular diseases. In the present study, the putative antihypercholesterolemic and antioxidative properties of an ethanolic extract of Piper betle and of its active constituent, eugenol, were evaluated in experimental hypercholesterolemia induced by a single intraperitoneal injection of Triton WR-1339 (300 mg/kg b.wt) in Wistar rats. Saline-treated hypercholesterolemic rats revealed significantly higher mean blood/serum levels of glucose, total cholesterol, triglycerides, low density and very low density lipoprotein cholesterol, and of serum hepatic marker enzymes; in addition, significantly lower mean serum levels of high density lipoprotein cholesterol and significantly lower mean activities of enzymatic antioxidants and nonenzymatic antioxidants were noted in hepatic tissue samples from saline-treated hypercholesterolemic rats, compared to controls. However, in hypercholesterolemic rats receiving the Piper betle extract (500 mg/kg b.wt) or eugenol (5 mg/kg b.wt) for seven days orally, all these parameters were significantly better than those in saline-treated hypercholesterolemic rats. The hypercholesterolemia-ameliorating effect was better defined in eugenol-treated than in Piper betle extract-treated rats, being as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt). These results suggest that eugenol, an active constituent of the Piper betle extract, possesses antihypercholesterolemic and other activities in experimental hypercholesterolemic Wistar rats.
PMCID: PMC3913032  PMID: 24523820
22.  Spectrum of Bacterial Keratitis at a Tertiary Eye Care Centre in India 
BioMed Research International  2013;2013:181564.
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.
PMCID: PMC3770006  PMID: 24066286
23.  Orthotopic neobladder perforation: an unusual presentation of small bowel obstruction 
Journal of Surgical Case Reports  2013;2013(7):rjt045.
Orthotopic bladder reconstruction is becoming increasingly popular in patients who have undergone radical cystectomy. One of the rare complications is spontaneous rupture, which presents with various symptoms, but in particular, abdominal pain. We report a case of orthotopic bladder perforation in a patient who presented with the symptoms and signs of small bowel obstruction.
PMCID: PMC3813458  PMID: 24964454
24.  Leaf evolution in Southern Hemisphere conifers tracks the angiosperm ecological radiation 
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.
PMCID: PMC3223667  PMID: 21653584
Podocarpaceae; molecular phylogeny; divergence time estimates; plant evolution
25.  Requirements for Efficient Correction of ΔF508 CFTR Revealed by Analyses of Evolved Sequences 
Cell  2012;148(1-2):164-174.
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.
PMCID: PMC3266553  PMID: 22265409

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