Characterizing compounds affecting ?F508 CFTR folding and conformation

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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Abstract
The F508 deletion in CFTR changes the proteins conformation, as a result of which it can no longer travel to the plasma membrane but is retained in the endoplasmic reticulum (ER). In search of new drugs to rescue the ?F508 phenotype many compound screens have been done and promising candidates have been identified, but their molecular mechanisms of action are still unknown. We developed an in vitro assay to follow conformational changes in CFTR and more specifically, in the F508-containing NBD1 domain. We subject in vitro translated radiolabeled NBD1 to limited proteolysis to examine conformational differences between wt and ?F508 NBD1. We clearly detect changes in conformation and are optimizing this assay further for examination of compound effects. To find out whether the screened compounds can affect NBD1 folding directly or indirectly we will add the drug at various stages of the limited proteolysis assay. Different compounds have been tested, including corrector 4a, which did not affect NBD1 folding. We concluded that these compounds do not rescue ?F508 CFTR by directly correcting NBD1, but must either affect CFTR domain assembly or act by changing the cell, for instance by activating a crucial chaperone

Author(s): Florence Peters, Elena Ganusova, Hanneke Hoelen, Mieko Otsu, Ineke Braakman

Implication of calnexin in the F508del-CFTR correction by miglustat

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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The most common mutation in cystic fibrosis (CF), F508del, results in CFTR (CF transmembrane conductance regulator) protein that is retained in the endoplasmic reticulum (ER). Previously, we have shown that miglustat corrects the defective trafficking of F508del-CFTR and hypothesized that by inhibiting the interaction of F508del-CFTR with calnexin, a lectin implicated in the ERQC (ER quality control), miglustat prevents the retention and the degradation of F508del-CFTR (Norez et al., 2006). However, others contest the role of calnexin in the F508del-CFTR retention (Okiyoneda et al., 2008). The purpose of the study was i) to determine the effect of small interfering RNA (siRNA) calnexin treatment on endogenous F508del-CFTR trafficking, ii) to compare these results with a miglustat induced-correction, iii) to understand whether calnexin is implicated in the F508del-CFTR trafficking and in its correction induced by miglustat. The human CF tracheal cell line CF-KM4 was transfected with a siRNA calnexin (0.5?g/mL, 72h), a siRNA control (0.5?g/mL) or was treated by miglustat (100?M, 2h). Then, the level of calnexin expression was tested by biochemical technique and consequences on CFTR and ENaC activities were assessed using single-cell fluorescence imaging. The results were compared with those obtained on untreated- and reverted- (CF-KM4 stably transfected with the CFTR wild type) CF-KM4 cells. We showed that decreasing calnexin expression (~ 75%) restores F508del-CFTR activity at the plasma membrane in correlation with a decrease (66%) of ENaC activity. Moreover, we found a 1.5 fold higher level of correction induced by miglustat than with siRNA calnexin : this level corresponds to the level of CFTR and ENaC activities measured in reverted CF-KM4. In conclusion, this work is in favor of a role of calnexin in the F508del-CFTR retention and confirms calnexin as a valuable CF therapeutic target. Nevertheless, our results also suggest that inhibition of calnexin/F508del-CFTR interaction is probably not solely sufficient to fully explain the effects of miglustat raising the hypothesis that another molecular target for this drug exists.

Author(s): Dorothee Raveau, Anne Cantereau, Frederic Becq, Caroline Norez

NEONATAL CORD BLOOD SCREENING WITH HPLC - TOWARDS COMPREHENSIVE AND IMPROVED PATIENT CARE OF SICKLE CELL DISEASE IN OMAn

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Feb 2017

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Abstract
Oman is a country with a population comprising of a wide range of ethnic groups, high rates of consanguinity and increased incidences of inter-cousin marriages. There is an increased prevalence of hemoglobinopathies which is of growing importance as knowledge of a population structure can be a unique aid in planning genetic services. The aim of this study was to establish neonatal cord blood screening in the Sultanate of Oman, in an effort to determine the prevalence of hemoglobinopathies by a costeffective method. High performance liquid chromatography [HPLC] is a powerful tool to screen newborns for hemoglobinopathies. Neonatal screening includes cord blood samples collection, screening, and follow up of all newborns with abnormal results. A total of 7837 consecutive cord blood samples were screened for presence of possible hemoglobinopathies by HPLC using Biorad Variant ?? program between April 2005 and March 2007. Complete blood counts [CBC] were also obtained on Cell Dyn 4000 automated blood cell counter. All samples were then processed to isolate and store mononuclear leukocytes for subsequent molecular diagnostics. The findings indicated a 47.07% incidence of ??thalassemia, based on low mean cell volume [MCV] and mean cell hemoglobin [MCH] on the CBC and significant amounts of Hb Barts on HPLC. The overall incidence of other hemoglobinopathies was 9.87%, with 5.47% incidence of sickle hemoglobin. On HPLC, D-window, E-window and C-window were present in 0.93%, 0.77% and 0.06% of the samples respectively. Since HPLC cannot diagnose beta thalassemia major at birth, in samples with HbA below 10%, the beta globin gene was directly sequenced including the promoter, all exons and introns in these samples. Amongst 206 (2.62%) samples sequenced, beta thalassemia trait was confirmed in 201 cases and 5 cases were found to be homozygous for beta thalassemia major. Additionally, direct sequencing of all abnormal samples with HbS [n=429], HbD [n=73], HbE [n=42], and HbC [n=5] was also performed on ABI Prism 3100 genetic analyzer to assign the correct genotype status to these subjects and use the same to validate the HPLC results. The significantly high incidence of hemoglobinopathies in newborns in the Sultanate of Oman emphasizes the value of neonatal cord blood screening to be implemented as the first step in the national strategy towards total management of hemoglobinopathies including early diagnosis, comprehensive clinical care and counseling of the affected families. The results of this large study wound indicate that using HPLC [<2 USD/sample] is a cost effective method. Moreover, prescreening of both parents and selecting only samples of neonatal cord blood from newborns with either parent having an underlying genetic trait for hemoglobinopathy would result in the a huge cost saving to the tune of over 90% as compared to universal neonatal cord blood screening and can be recommended as a highly cost-effective method targeted to screen only the abnormal samples.

Author(s): Al-Kindi S., Pathare A.V., Al-Madhani A., Al-Zadjali S., Krishnamoorthy R.

THREE OBSERVATIONS OF CONTINUAL SKIN PEELING SYNDROME

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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Abstract
The continual skin peeling is a rare icthyosis. However several cases have been reported in the south bank of the Mediterranean Sea (Strong endogamia hot climate). We report 3 male patients born to case at the age of five and later in the two others at the age of ten. All the 3 patients have the same predisposing factors (summer, heat, sweat, friction). In one case, the efficiency of acitretine at a 60 mg dose per day resulted in improvement.

Author(s): Stambouli O.B.

SERVICE INDICATORS FOR A REGIONAL HEMOGLOBINOPATHY PREVENTIVE PROGRAM IN DOHUK-IRAQ

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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Abstract
?-Thalassemia major and sickle cell disease are important health problems in Dohuk Governorate at the extreme north of Iraq, which made instituting proper preventive programs a necessity. Accordingly, and as a prerequisite for such a program, the Governorate was mapped for these hemoglobin disorders, the service indicators of the preventive program were assessed, and the ?-thalassemia mutations were characterized. A total of 591 couples (1182 individuals) attending the health centers for premarital checks were screened. 44 (3.7%) were found to be carriers of ?-thalassemia, 14 (1.2%) of the sickle cell gene, and one (0.1%) of ??-thalassemia. Three couples (5 / 1000) were found to be at risk of having children affected by ?-thalassemia major. While the estimated number of affected children expected with a major hemoglobinopathy was 39 per year. The ?-thalassemia defects of 213 chromosomes were characterized using ARMS and reverse hybridization techniques and it was found that the most prevalent mutation was the IVSII.1 (G>A; 22.1%), followed by Codon 44 (-C; 14.1%) and IVS I.1 (G>A; 10.3%). The above findings stress the importance of a regional preventive program, and will help set the stage for initiating such a program for these hemoglobinopathies based on premarital screening, counseling and prenatal diagnosis.

Author(s): Al-Allawi N., Jubrael J., Anwar A., Fariq F.

Large-Scale Western-Blots to Assess Effects of siRNAs on CFTR Trafficking

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Jan 2017

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CFTR biogenesis starts with the co-translational folding and insertion into the endoplasmic reticulum (ER) membrane, followed by core-glycosylation and exit to the Golgi. CFTR glycan moieties are then processed along the Golgi, generating the mature membrane-resident form. This process is known as processing or maturation and a simple Western blot (WB) analysis distinguishes the core-glycosylated immature ER-specific form of CFTR (band B) from its fully-glycosylated mature post-ER form (band C). This biochemical assay is thus used to monitor CFTR exit from the ER. Moreover, as it provides quantitative information on steady-state levels, it can be used to assess the impact on CFTR of chemicals or molecules (over or underexpression of a given gene). Our goal was to optimize WB to establish a robust, simple and sensitive method to analyse CFTR expressed in cell lines (epithelial and non-epithelial) grown on 96-well plates which can be used as a secondary test after high-content siRNA screen for proteins involved in CFTR trafficking. Different cell lines were tested here: Calu-3, CFBE and BHK stably expressing either wt- or F508del-CFTR. WB was performed as previously but using benzonase to shear DNA. In each lane of a 7% (w/v) SDS-PAGE gel, contents of 1 to 6 wells from a 96-well plate were applied (about 7.5 mg to 45 mg protein). After electrophoresis and electroblotting, the membrane was blocked and incubated with the 596 anti-CFTR antibody (CFF) at 1:1000, followed by incubation with secondary antibody. Detection was done using West Pico System (Pierce). First, we determined the minimal amount of cells needed to detect CFTR by WB and we found that ~4 x 104 cells (number present in one well) is enough to detect both bands B and C of CFTR in the above cells lines. We then determined the sensitivity of this assay to effects caused by siRNA transfection by using as control siRNAs against CFTR (Silencer Select, Ambion, ref. s2945, s2947). We found a significant decrease (~65 and ~85% respectively) in the amounts of band B and C for wt-CFTR, after 48h of transfection with 1.2 pmol of siRNA. We conclude that this large-scale WB assay is robust to test the effect of different siRNAs against genes possibly affecting the trafficking of wt- and F508del-CFTR.

Author(s): Marta A. Palma, Carlos M Farinha, Margarida D. Amaral

THE IMPACT OF GENOME INFORMATICS ON NEXT-GENERATION PERSONALIZED DIAGNOSTICS AND MEDICINES

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Feb 2017

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Abstract
The advent of 2nd generation, low-cost, DNA sequencing technologies is leading to dramatic increases in the rate of genome data generation. The utilization of these new technologies to sequence ever increasing numbers of human genomes for personalized diagnostics and medicines will generate even more data. Current database and software technologies are becoming progressively ineffective for managing, processing and analyzing these new levels of information. Synamatix addresses this need by leveraging SynaBASE?, which is an innovative database solution based upon indexing patterns in data. Rapid cross referencing of clinical patient sequence data back to the human genome is a critical step in SNP discovery and identifying potential disease states. Performance improvements of between 100 and 10,000 fold for comparative and differential genomics and for mapping-based genome assembly from 2nd generation sequencers will be reviewed.

Author(s): Anwar A.

Interaction between CFTR and Genistein at different values of intracellular pH

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Feb 2017

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Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are compounds with a double effect. At lower concentration they increase the activity of CFTR, whereas at higher concentrations they inhibit it. The binding site for potentiators is hypothesized to be located in the interface of the dimer formed by the two nucleotide binding domains (NBDs), interacting mostly with NBD1 and to a lesser extent with NBD2. This binding site seems to involve at least two protonable residues, Cys491 and His1348. The binding of potentiators to the NBDs probably relies on electrostatic interaction, as electrostatic interactions between oppositely charged amino acids might be involved in the formation and stabilization of the dimer. Here we have analysed the electrostatic contribution to the binding of genistein by modifying intracellular pH. We have analysed the effect of pH on both, the activating and the inhibitory dissociation constants. The study was done on polarized epithelia expressing high levels of wild type CFTR after establishing that modifications in the pH of the basolateral solution, between pH 6 and 8, change the intracellular pH to the same extent. We found that at pH 6 it was necessary a lower concentration of CPTcAMP to activate CFTR. In addition, we found that the maximal CFTR current was significantly lower at this pH value. Regarding genistein binding, we found that at alkaline pH the apparent dissociation constant for the activating site was shifted to higher concentrations. In contrast, we found that the inhibitory apparent dissociation constant was affected by alkaline and acidic pH values to the same extent. In fact, at pH 6 and at pH 8 the affinity of genistein for the inhibitory site was similarly increased. This study suggests that cysteine, possibly Cys491, is involved in the activating binding site. In contrast, an histidine (e.g. His1348) does not seem to be part of it. Besides, our data indicate that the inhibitory site, whose location is still unknown, may contain both, a cysteine and a histidine, the only two amino acids with a pKa in the range of the pH analysed. This work was supported by the Italian Cystic Fibrosis Foundation grant FFC#2/2008 with the contribution of Mille bambini a Via Margutta onlus, Blunotte, and Lega Italiana FC - Associazione Toscana.

Author(s): Raffaella Melani, Olga Zegarra - Moran

Effect of Cigarette Smoke Extract on IL-8 Release from Primary Nasal Epithelial Cells in CF and Healthy Volunteers

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Feb 2017

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Cigarette smoke extract (CSE) decreases the expression of the CFTR. Furthermore, CSE has profound effects on epithelial cells but different results are reported with cell lines and primary cells. This study aimed to compare the effects of CSE on spontaneous and induced IL-8 release from primary nasal epithelial cells isolated from CF patients possessing two different classes of CFTR mutation (F508del homozygous; R1 17H heterozygote) and healthy volunteers. Nasal epithelial cells were obtained via nasal brushings of the inferior turbinate of CF patients; F508del homozygote (n=5), R1 17H heterozygote (n=7) and healthy volunteers (n=6). The cells were expanded in culture and used at passage 3. Monolayer cultures were pre-exposed for 4h in the presence or absence of 5% CSE. The cells were then stimulated with LPS from P. aeruginosa (LPS-PA 50 or 100 ?g/ml); Cytomix 1 (TNF-? 10 ng/ml, IL-1? 5 ng/ml, LPS-PA 5 ?g/ml) or Cytomix 2 (TNF-? 10 ng/ml, IL-1? 10 ng/ml, IFN-? 1000 U/ml) for 24 h. Culture supernatants were harvested and IL-8 release measured using ELISA (R & D systems). LPS (100 ?g/ml) induced IL-8 release in control cells was increased by CSE (1188?292 vs. 1774?501.6, p< 0.05). In cells from F508del homozygous patients CSE increased cytomix 2 and LPS 50 ?g/ml induced IL-8 secretion. In the R1 17H heterozygotes only the spontaneous IL-8 secretion was significantly increased by CSE exposure. Comparing the responses from the various genetic backgrounds of the subjects; the only significant differences between the groups were with cytomix 2 where the releases followed R1 17H< F508del< control (8157?1746, vs 16031?3729 vs 21296?2576). R1 17H cells were also significantly less responsive to cytomix 2 compared to F508del cells in the presence of CSE.In contrast to our data using cell lines, where CSE consistently decreased IL-8 secretion from the CF cell line (CFBE41o-), preincubation of primary cells with CSE only caused sporadic increases in IL-8 release. Cytomix 2 induced significantly different levels of IL-8 secretion depending on the genetic background of the cells. As yet, we have no explanation for these data but further studies will investigate the action of IFN-? alone on primary cells. Williams MTS et al. Ped Pulmon 2008;287:246

Author(s): Mark Thomas Shaw Williams, Madeleine Ennis, Joseph Stuart Elborn

STUDY OF CYP2D6 POLYMORPHISM IN PARKINSONS AND CONTROL SUBJECTS FROM INDIA

Medicine Sciences and Healthcare Journal (MSHJ), Volume 2, Feb 2017

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Abstract
Parkinsons disease (PD) is the second most common neurodegenerative disorder next to Alzheimers disease. It accounts for about 0.2 to 1% of the age of 65 ? 69 and raises to 1 ? 2% of the age of 80 and above. It is mainly characterised by the involvement of the four classical symptoms - tremor, bradykinesia, postural instability and the rigidity. It may be due to the cause of the environmental toxins and also due to the genetic factors. There is high relationship for the PD with the environmental toxins. The enzyme CYP2D6 is one which acts on the chemical 1-methyl-4-phenyll, 2,3,6- tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and many other neurotoxins that cause the PD. An exciting possibility is that chemical transformation to produce PD toxin in the course of the detoxification mediated by CYP2D (cytochrome P450). The CYP2D comprises many isoenzymes which are involved in the metabolism of exogenous and endogenous toxins. The CYP2D subfamily comprise of genes which are highly specific for the debrisoquine- 4 hydroxylase activity. In this, the CYP2D6 is widely studied polymorphism. In the present study, we analyzed this CYP2D6*4 polymorphism in both the PD cases and controls using polymerase chain reaction (PCR) and Restriction Fragment Length polymorphism (RFLP). Bearers of the CYP2D6 gene variants may be classified as the extensive metabolizers (EM) and poor metabolizers (PM). PM allele carriers have lower gene activity and they are more prone to the environmental toxins. In this study we determined the CYP2D6*4 allele in both PD and control to evaluate the relation of this allele with the PD.

Author(s): Padmaja M.V., Kifayathullah L., Srinivasan A.V., Ramesh A.