Whole Exome Re-Sequencing Implicates CCDC38 and Cilia Structure and Function in Resistance to Smoking Related Airflow Obstruction

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV₁) despite many years of heavy smoking. We hypothesised that these “resistant smokers” may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the “resistant smokers” and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34×10⁻⁴) in CCDC38, a gene which has previously been reported to show association with FEV₁/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.     

Expeditious synthesis and chromatographic resolution of (+)- and (-)-trans-1-benzylcyclohexan-1,2-diamine hydrochlorides

The hitherto unknown (-)- and (+)-1-benzylcyclohexan-1,2-diamine hydrochlorides 4a. HCl and 4b. HCl were synthesized by means of diastereoselective alpha-iminoamine rearrangement with subsequent imine reduction and hydrogenolysis. The relative trans-configuration as well as the absolute (1S,2R) and (1R,2S) configurations of 4a and 4b, respectively, were elucidated on the basis of an X-ray analysis of 3b. HCl. The enantiomeric excess (ee) values of the title compounds (>99%) were determined by chiral HPLC on a Chirex (D) Penicillamine column.     

Synthesis of biodegradable emulsifier using lipase in anhydrous pyridine

Summary Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Rewinding the tape: selection of algae adapted to high CO2 at current and pleistocene levels of CO2

Selective history is thought to constrain the extent and direction of future adaptation by limiting access to genotypes that are advantageous in a novel environment. Populations of Chlamydomonas previously selected at high CO2 were either backselected at ambient levels of CO2, or selected at levels of CO2 that last occurred during glaciation in the Pleistocene. There was no effect of selective history on adaptation to either level of CO2, and the high CO2 phenotypes were evolutionarily reversible such that fitness in ambient CO2 returned to values seen in controls. CO2 uptake affinity improved relative to the ancestor in both ambient and glacial CO2, although wild-type regulation of CO2 uptake, which deteriorated during previous selection at high CO2, was not restored by selection at lower levels of CO2. Trade-offs in both CO2 uptake affinity and growth were seen after selection at any given level of CO2. Adaptation to ambient and glacial-era levels of CO2 produced a range of phenotypes, suggesting that chance rather than selective history contributes to the divergence of replicate populations in this system.     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

Treatment of odorous volatile fatty acids using a biotrickling filter

In this study, a novel fibrous bioreactor was developed for treating odorous compounds present in contaminated air. The first stage of this work was a preliminary study which aimed at investigating the feasibility of using the fibrous bioreactor for the removal of malodorous volatile fatty acids (VFA) that is a common odorous contaminant generated from anaerobic degradation of organic compounds. The kinetics of microbial growth and VFA degradation in the selected culture, and the performance of the submerged bioreactor at different VFA mass loadings were studied. Above 95% of VFA removal efficiencies were achieved at mass loadings up to 22.4 g/m(3)/h. In the second stage, the odour treatment process was scaled up with system design and operational considerations. A trickling biofilter with synthetic fibrous packing medium was employed. The effects of inlet VFA concentration and empty bed retention time (EBRT) on the process performance were investigated. The bioreactor was effective in removing VFA at mass loadings up to 32 g/m(3)/h, beyond which VFA started to accumulate in the recirculation liquid, indicating the biofilm was unable to degrade all of the VFA introduced. Although VFA accumulated in the liquid phase, the removal efficiency remained above 99%. This suggested that the biochemical reaction rather than gas-liquid mass transfer was the limiting step of the treatment process. In addition, the biotrickling filter was stable for long-term operation with relatively low and steady pressure drop, no clogging and degeneration of the packing material occurred during the four-month study.